Aus dem Institut für Anatomie
Geschäftsleitung: Prof. Dr. P. Gehr
Arbeit unter der Leitung von
Dr. sc. nat. ETH D. Studer und Prof. Dr. med. P. Eggli
A newly designed specimen preparation system
allows fast sampling of blebbing cells prior to
high pressure freezing: new insights in the
mechanism of bleb protrusion
Inaugural-Dissertation
zur Erlangung der Doktorwürde der Philosophie
im Fach
Strukturbiologie
vorgelegt von
Dimitri Vanhecke
Aus Brugge
Koninkrijk België
Von der Medizinischen Fakultät der
Universität Bern
auf Antrag der Dissertationskommission
als Dissertation genehmigt
Promotionsdatum:
Der Dekan der Medizinischen Fakultät
Contents
Contents
Contents ..............................................................................................................I
List of abbreviations ............................................................................................. 1
Abstract.............................................................................................................. 2
Zusammenfassung ............................................................................................... 3
1. Aim of the Study .............................................................................................. 4
2. Introduction..................................................................................................... 5
2.1. Cellular motility .......................................................................................... 5
2.1.1 Definition ............................................................................................. 5
2.1.2 Phenotype of Walker carcinosarcoma cells................................................. 5
2.1.3 General locomotion model ...................................................................... 6
2.1.4 Force generation of Blebbing ................................................................... 8
2.2. Biological electron microscopy .................................................................... 13
2.2.1 Electron microscopy and related problems............................................... 13
2.2.2 Alternatives to chemical fixation ............................................................ 13
2.2.3 Cryotechniques ................................................................................... 14
2.2.4. High pressure freezing artifacts ............................................................ 19
2.3. Fast Sampling for High pressure freezing ..................................................... 21
2.4. The state of water and ions in the cell ......................................................... 22
2.4.1 Cell water........................................................................................... 22
2.4.2 Ling’s fixed charge hypothesis ............................................................... 22
2.4.3 Salt linkage ........................................................................................ 24
3. Results.......................................................................................................... 25
3.1. Published results ...................................................................................... 25
3.2. Manuscript in preparation: Hitherto not described inclusions in the front of Walker
carcinosarcoma cells may play a role in cellular motility........................................ 26
3.3. Additional results...................................................................................... 49
3.3.1 Dextran toxicity................................................................................... 49
3.3.2 The microbiopsy system ....................................................................... 50
3.3.3 Alternative sampling of cell suspensions.................................................. 51
4. References .................................................................................................... 53
5. Appendix....................................................................................................... 62
Appendix 1 .................................................................................................... 62
Appendix 2 .................................................................................................... 62
6. List of publications .......................................................................................... 63
Peer reviewed articles .................................................................................. 63
I
Contents
Book chapters ............................................................................................. 63
In preparation ............................................................................................. 63
Proceedings ................................................................................................ 63
7. Curriculum vitae............................................................................................. 64
8. Acknowledgements ......................................................................................... 65
II
List of Abbreviations
List of abbreviations
DIC: Differential interference contrast
EC: extracellular (cfr. IC)
FD: Freeze-drying
FS: Freeze substitution
HPF: high pressure freezing
IC: Intracellular
LM: Light microscopy
MT: microtubuli
RT: Room temperature
TEM: transmission electron microscope
WCS: Walker carcinosarcoma cells
1
Abstract
Abstract
A specimen preparation system was developed allowing fast and gentle transfer of
samples to a Leica EMPACT high-pressure freezer (Leica, Vienna, Austria). This technical
breakthrough (commercialised by Leica, Vienna, Austria) made it possible to prepare a
sample faster before cryo-immobilization, hence closer to its native structure. Using this
method, the ultrastructure of Mammalian cells was improved compared to that of
conventionally fixed specimens.
A large frontal inclusion termed pseudovacuole is described in the non-adherent
metazoan Walker carcinosarcoma cell line. Differential interference contrast (DIC) and LM
observations of living Walker carcinosarcoma cells showed an entity at the front of the
cell, typically in the neighbourhood of blebs. The inclusion is vacuole-like, but fluorescent
dyes, as well as transmission electron microscopy failed to show a surrounding
membrane, hence the name pseudovacuole. Fluorescent trackers demonstrated very high
abundance of the highly basic amino acid lysine, while markers for nucleic acids and
lipophylic regions revealed the pseudovacuoles as a lipid and nucleic acid-free
cytoplasmic region.
Transmission electron microscopic (TEM) micrographs showed unstained apparently
empty zones in chemical fixed Walker carcinosarcoma cells. Our newly designed sampling
system allowed sampling before cryofixation of cell pellets in less than 30 seconds. After
cryofixation by high pressure freezing (HPF) followed by freeze substitution (FS) and
resin embedding, TEM micrographs revealed similar zones as seen in chemical fixed cells.
However, the zones observed in high pressure frozen samples are not empty. Although
faintly stained, a loose degree of organization, unrelated to membranes, is recognizable.
Cell organelles and ribosomes were not found in these zones. Light scanning microscopy
(LSM) of lysine tracker stained cells embedded in Epon shows a co-localisation between
the pseudovacuoles observed in DIC and fluorescence images, and the faintly stained
zones observed in the TEM. Therefore I postulate that these faintly stained zones in TEM
are pseudovacuoles.
We can only hypothesize about the function of the pseudovacuoles. Its location,
hydrophilic content, dynamics and behavior point in the direction of a possible
involvement in bleb formation, i.e. as an osmotic tool regulating intracellular pressure.
The data gathered suggests this entity would act according to Ling’s fixed charge
hypothesis.
2
Zusammenfassung
Zusammenfassung
In der nicht-adhärenten metazoanen Zelllinie Walker carcinosarcoma Zelle ist ein grosser
frontaler Einschluss, eine so genannte Pseudovakuole, gefunden worden. Mit der Methode
des
differentiellen
Interferenz
Kontrasts
(DIC)
ist
an
der
lebendigen
Walker
carcinosarcoma Zelle eine Einheit sichtbar, die sich vorne befindet, meistens in der Nähe
eines Blebs. Der Einschluss ist Vakuolen-artig aber mit fluoreszierenden Färbung oder
Transmissions-Elektronenmikroskopie ist keine Membran zu erkennen, die diese Einheit
umgibt, deshalb auch der Name Pseudovakuole. Fluoreszierende Marker zeigen eine
grosse Fülle an sehr hydrophilen Aminosäure-Lysine; behandelt man die Pseudovakuole
jedoch mit Markern für Nukleinsäuren und hydrophobe Umgebungen erscheint sie als
Lipide und nukleinsäurefreie, cytoplastische Zone.
Es wurde ein spezifisches System entwickelt, das einen schnellen und schonenden
Transfer von Proben in den Leica EMPACT Hochdruck-Gefrierer (Leica, Wien, Österreich)
ermöglicht. Diese technische Innovation (von Leica kommerzialisiert) erlaubt es, Proben
schneller zu präparieren bevor sie Cryo-immobilisiert werden, so dass die ursprüngliche
Struktur besser erhalten bleibt.
In Transmissionelektronenmikroskopischen Aufnahmen (TEM) erscheinen Zonen in
chemisch fixierten Walker carcinosarcoma Zellen ohne Kontrast, die scheinbar leer sind.
Mit dem neu entwickelten System wurde Sampling vor der Kryofixation in weniger als 30
Sekunden möglich. TEM-Aufnahmen nach einer Hochdruck-Kryofixation (HPF), gefolgt
von einer Kryosubstitution (FS) und Plastikeinbettung, enthüllten ähnliche Zonen wie sie
schon in chemisch präparierten Zellen gefunden wurden. Diese Gebiete der in Hochdruck
gefrorenen Zellen sind jedoch nicht leer. Obwohl sie nur leicht kontrastiert sind, ist eine
schwache Organisation erkennbar, jedoch ohne Bezug zu Membranen. In diesen Zonen
wurden keine Zell-Organellen oder Ribosomen gefunden. Licht Raster Mikroskopie (LSM)
von mit Lysin-Tracker behandelten und in Epon eingebetteten Zellen zeigt eine KoLocalisation der mit DIC oder Fluoreszenz beobachteten Pseudovakuolen, und den
schwach kontrastierten Gebieten, die mit TEM gesehen wurden. Wir postulieren deshalb,
dass es sich bei diesen schwach konstratierten
Zonen
in
TEM-Aufnahmen
um
Pseudovakuolen handelt.
Über die Funktion dieser Pseudovakuolen können wir nur Hypothesen aufstellen.
Aufgrund ihrer Lage, des hydrophilen Inneren, ihrer Dynamik und des Verhaltens ist es
wahrscheinlich, dass sie in die Bleb-Bildung involviert sind. Eine mögliche Funktion ist die
intrazelluläre osmotische Druckregulation. Die bisher gesammelten Daten deuten darauf
hin, dass sich diese Pseudovakuolen übereinstimmend mit „Ling’s fixed-charge“
Hypothesen verhalten.
3
Aim of the study
1. Aim of the Study
The shape of living cells is determined by the cortex and cortical actin contraction but
also by hydrostatic pressure. Cells tend to shrink and develop major artefacts following
standard chemical fixation. Particularly the effects of hydrostatic pressure are easily
distorted by chemical fixation. However, it might be of utmost importance to have the
original structure of the cells as exhibited during locomotion to determine the original
basis of locomotion.
“Vacuoles” have been observed at the front of spontaneously blebbing Walker
carcinosarcoma cells, but they become very prominent in blebbing cells. These structures
are not sufficiently preserved using standard fixation techniques.
The initial objective of the study was the characterisation of locomoting cells using
alternative fixation methods, i.e. cryomethods. A way had to be found to sample the
living state of the cells and not a stressed state. Fast sampling methods and new
technical developments were worked out allowing the preparation of not only cell
suspensions, but also tissue samples.
Early findings confirmed the existence of the “vacuole” introduced above, whose
structure and content was preserved as well. The study developed towards the structural
characterisation of this entity, which I termed pseudovacuole, using light, confocal and
electron microscopy and the characterisation of its content.
4
Introduction
2. Introduction
2.1. CELLULAR MOTILITY
2.1.1 DEFINITION
Concerning motility, several terms are in use throughout the literature. Motility (noun
from “motile”) is described by the Henderson’s dictionary of biological terms (11th ed.)
as “capable of spontaneous movement”, which includes change of place, position and
posture. Hence, locomotion (syn. motivity), blebbing, phagocytosis, contractile vacuoles,
et cetera are all examples of motility. The presented work focuses on blebbing.
2.1.2 PHENOTYPE OF WALKER CARCINOSARCOMA CELLS
Walker carcinosarcoma cells (WCS) were originally obtained from a spontaneous
carcinoma of the mammary gland of a pregnant albino rat. WCS resemble a carcinoma in
young transplants and a sarcoma in older transplants (Stedman’s 25th edition). The cells
exhibit a non-adherent state (subline 1) but adhere by means of focal adhesions to the
culture flask (Subline 2) when cultivated over longer periods (> 2 weeks). When
restricted between coverslips, Subline 1 establishes a significantly higher locomotive
speed than subline 2 (Sroka et al., 2002). All experiments were carried out with subline 1
cells in suspension.
Figure 1 shows the essential characteristics of a Walker carcinosarcoma subline 1 cell as
a scheme (left) and in DIC picture (right). At the front of the cell, three types of
protrusions are established: lamellipodia, filopodia and blebs.
Filopodia: the simplest form of protrusions. Filopodia are finger-like cylinders enclosing
tight bundles of actin filaments, all oriented in the direction of the protrusion (Sheetz et
al., 1992).
Lamellipodia: flattened sheets containing an organised α-actinin bound (Kole et al.,
2005) orthogonal cross-weave between two sets of actin filaments (Small et al., 1999)
Blebs1: typically shaped as a hemispherical cap. The cortex around these structures is
formed of a much less organised actin mesh (Cox, 1995, Boulbitch et al., 2000).
Organelles are absent in the frontal area, giving it a smooth hyaline appearance in LM,
while the cell body contains the nucleus and the organelles (e.g. vesicles and
mitochondria) resulting in a granular appearance (granuloplasm). The pseudovacuole is a
large inclusion in the frontal, hyaline cytoplasm. A backwards extension, termed uropod,
1
Blebs are a type of lobopodia, which is defined as “hemispherical protrusions with a much less ordered actin
cortex distribution” (Boulbitch et al., 2000). Lobopodia include blebs and pseudopodia, and are found
throughout animal phylogeny: Amoeba sp., Xenopus eggs, Neuroblastoma cells, granulocytes, Walker
carcinosarcoma cells, etc.
5
Introduction
is present in polarized cells (70 to 90% of the cells under optimal conditions, Sroka et al.,
2002, Keller and Eggli, 1998a). When a uropod is not present, the cell is considered as
not polarized, which is reflected by a spherical appearance. However, also spherical cells
can still bear protrusions (Figure 1, DIC picture, right cell).
front
cell body
L Hc
F
P
B
V
rear
Gc
M
Hc
Gc
N
Mt
A
B
L
U
My
B
U
Figure 1. Left: scheme of a polarized Walker carcinosarcoma cell. The cell is opened to
allow a view on the interior. Right: DIC picture of a polarized (left) and a spherical cell
(right) with protrusions. Bar= 10 µm. Abbreviations: A= actin cortex*, B= bleb, F=
filopodium*, Gc= granular cytoplasm, Hc= hyaline cytoplasm, L= lamellipodium, M=
mitochondrion*, Mt= microtubuli*, My= cortical myosin*, N= nucleus*, P=
pseudovacuole*, U= uropod, V= vesicle*. * Not shown in DIC picture.
2.1.3 GENERAL LOCOMOTION MODEL
Since the publication of the first model for amoeboid movement by Mast (1926), a
variety of cell types was used as model systems for motility and locomotive studies:
fibroblasts, fish keratocytes, leucocytes, Amoeba proteus, Dictyostelium discoideum,
Walker carcinosarcoma cells, Xenopus oocytes, PtK2 cells infected with Listeria
monocytogenes virus, etc. Despite almost 80 years of intensive research, a unified model
explaining the driving force of motile functions throughout the Eukaryotic group could not
be distilled. The possibility that multiple ways of force generation of motile functions
might have evolved should be kept in mind, thus rendering a unified model impossible.
However, the picture of locomotion in tissue cells is getting sharper. The process can be
subdivided in four different successive manifestations of motility (Mitchison and Cramer,
1996; Heidemann and Buxbaum, 1998, Sheetz et al., 1999).
Protrusion: The forward movement of the membrane at the front of the cell, by filopodia,
lamellipodia or blebs.
Adhesion: The connection between the leading front and the substrate by means of focal
adhesions. Adhesion is required for protrusion to be converted into movement along a
substrate.
Traction: The process of forward movement of the cell body.
6
Introduction
Deadhesion and tail retraction: Two distinct mechanical processes, which, depending on
the cell type, take place in a more or less efficient manner, or not at all (e.g. neuronal
growth cones lack tail retraction).
Tissue cells of various morphologies usually form lamellipodia. Locomotive functions are
controlled through regulation of actin dynamics (polymerisation and sliding) in the
lamellipodium. Therefore, the lamellipodium is the primary locomotory organelle in tissue
cells (Keller et al., 2002a). Rapidly moving cell types (with poor adhesion abilities), such
as Amoeba proteus and WCS, tend to use blebbing as their main mode of locomotion
(Peckham et al., 2001).
Tissue cells
Theoretically, the force for protrusion can be generated either by frontal (local) actin
polymerisation, by actin polymerisation in the cell body (and transmitted to the front by
mechanical linkage) or by pressure. Forward movement of actin has not been observed,
and a bulk hydrostatic pressure seems absent (Mitchison and Cramer, 1996). It is
thought that local actin polymerisation in the front of the cell is the motor for protrusion
(Abercrombie et al., 1970a, b, c, 1971, 1972, Lee et al., 1993) and incorporation of actin
subunits takes place at the distal tip of the protrusion (Wang, 1985). According to the
model of Zhu and Skalak, (1988), the actin filament elongation is carrying out
mechanical work against the opposing pressure of the cell membrane. As a result,
pressure drops at the tip of the protrusion, creating a pressure gradient that drives a
fluid stream, which in turn brings more actin monomers to the growing tip.
Adhesion is accomplished via transmembrane proteins (integrins) on the cell surface that
link the cortical actin cytoskeleton (Bretscher, 1996, Anderson and Cross, 2000, WehrleHaller and Imhof, 2002) with the extracellular space. Incorporated actin in the cortex is
continuously moving towards the rear – in the reference frame of the cell. The rearward
movement of actin filaments is balanced by filament assembly at the front of the cell and
depolarisation towards the rear (Figure 2).
During keratocyte traction, compression is exerted perpendicular to the direction of
locomotion (Oliver et al., 1995, Oliver et al., 1999). The cell body is not passively sliding
behind the leading edge, but rolls forward (Anderson et al., 1996) due to inward
retraction of the margins (Burton et al., 1999) generated by actomyosin depending
contraction between the lamellipodium and the cell body (Paku et al., 2003). In fish
keratocytes cell body rolling was estimated to be responsible for about 50% of the
distance travelled, while the remainder is due to sliding (Svitkina et al., 1997).
Affinity modulation of integrin-ligand interactions in the focal contacts reduces the
adhesion strength in the rear of the cell (Friedl and Bröcker, 2000). Moreover, myosin II
activity in the rear (Rafelski and Theriot, 2004) pulls the focal adhesions away from the
substrate. Fibroblasts are found to leave behind a trail of cytoplasmic fragments in vitro,
7
Introduction
loosing a considerable fraction of the adhesion molecules as they move (Lauffenburger
and Horwitz, 1996). On the other hand, weakly adhesive and fast moving cells (e.g.
leukocytes) carry out this step more efficiently and adhesion molecules are recycled
through dispersion over the cell membrane or via endocytic vesicles (Palecek et al.,
1996).
Cell body rolling
Lamellipodium
Actin depolymerisation site
Actin
polymerisation
site
Nucleus
Substratum
g-actin
Cell body sliding
Transmembrane adhesion protein
Adhesion molecule recycling
Figure 2. Actin cycle based locomotion mechanism of tissue cells. Actin polymerisation at
the leading edge is pushing the cell front forwards. Attachment to the substrate allows
traction of the cell body via sliding and rolling. Actin depolymerisation in the rear is
generating g-actin. Molecules involved in focal contacts can be recycled via endocytosis.
Yellow arrows: recycling of actin, green arrows: recycling of adhesion molecules via
vesicle trafficking. The cell is moving to the left
Rapidly moving and invasive cells
It is understood that the former model explains the basic locomotive forces in normal
tissue cells, but it has difficulties explaining bleb and pseudopod motility in rapidly
moving, invasive cell types (Peckham et al, 2001). Actin polymerisation is not needed for
the formation of blebs (Cunningham, 1995, Keller and Eggli, 1998a, Hagmann et al.,
1999, Stossel et al., 1999, Boulbitch et al., 2000, Keller, 2000), and drug-treated Walker
carcinosarcoma cells lacking frontal actin (for details, see further) locomote faster than
untreated cells (Keller et al., 2002a). Furthermore, it has been measured that blebs
expand faster than actin can polymerise (Janson and Taylor, 1993, Keller and Bebie,
1996). Clearly, other factors than actin polymerisation must be involved in these events.
2.1.4 FORCE GENERATION OF BLEBBING
Micropipette experiments
After microsurgical destruction of the frontal zone of Amoeba proteus the cytoplasm was
squeezed through the breach into the external medium (Grebecka and Grebecki, 1981).
It was found that the intracellular hydrostatic pressure rises significant shortly before the
onset of pseudopod formation (Yanai et al., 1996). Induction of a sudden increase in
intracellular hydrostatic pressure by deformation of entire Walker carcinosarcoma cells
8
Introduction
into a sausage-like shape by aspiration into a micropipette (inner diameter 10 µm) led to
the formation of blebs and a hyaline cap, while the formation of lamellipodia was not
observed (Schütz and Keller, 1998). After release, the cells reacquired their initial shape
and protrusions (either spherical or polarized, bearing blebs and/or lamellipodia). Suction
pressure applied through smaller micropipettes (inner diameter 5 µm), induced separate
blebs, and moreover, the blebs persisted (Rentsch and Keller, 2000). These experiments
show that pressure changes are an essential initial factor in bleb formation.
Tonicity experiments
Although assigning great importance to the actin cytoskeleton to explain locomotive
motility, Dipasquale (1975) recognised the role of internal hydrostatic pressure in the
formation of blebs. Blebbing ceased in marginal epithelial cells when increasing the
extracellular tonicity but was regained upon restoring the initial tonicity. Furthermore,
hypotonic media increased blebbing activity, a treatment shown to be reversible as well
(Dipasquale,
1975).
Hypotonic
media
increased
displacement
efficiency
of
fish
keratinocytes 2.5 times (Korohoda et al., 1997) while hypertonic media completely
inhibited migration of smooth muscle cells (Schousboe, 2003). Apparently, extracellular
hypertonicity is reducing motile functions, while extracellular hypotonicity is enhancing
them.
Drug experiments
In attempts to reveal the pathways involved in bleb motility, blebbing cells were
subjected to a variety of drugs (summarized in Table 1).
Due to interaction between the actin cytoskeleton and microtubuli (Jung et al., 1997,
Waterman-Storer
and
Salmon,
1999)
cytoskeleton
acting
drugs
have
complex
consequences. Treatment with MT binding agents (colchicine, 10-5M) results in the
detachment of the actin cortex in the front of Walker carcinosarcoma cells, thereby
developing blebs constantly (Keller and Eggli, 1998a, Keller, 2000). Treatment with an
actin polymerisation inhibitor (Latrunculin A, 10-8M to 10-7M) caused immediate change
of type of protrusion from lamellipodia to blebs (Keller, 2000). Moreover, combined
treatment of these drugs resulted in a localised depletion of actin in the front of the cell
and an increased locomotion speed compared to untreated cells (Keller et al., 2002a, b).
Figure 3 shows the essential part of the Rho A activated actomyosin depending
contraction pathway for cell motility. Staurosporine (Thuret et al., 2005), an ATP
analogue blocking kinases thereby completely prevented blebbing in NB2a cells. Calyculin
A treatment – inhibiting the activity of phosphatases (Gu et al., 2003) – resulted in very
intensive blebbing. In addition, also lysophosphatidic acid (LPA) induced immediate
blebbing in NB2a cells upon administering in the medium (Hagmann et al., 1999).
9
Introduction
Y-27632 (Uehata et al., 2000), an agent specifically blocking Rho-activating kinase
(ROCK)
suppresses
blebbing
and
inhibits
polarity
and
locomotion
of
Walker
carcinosarcoma cells (Keller et al., 2002a, Wicki and Niggli, 2001). KT5926 (Nakanishi et
al., 1990), an inhibitor of myosin light chain kinase (MLCK) blocked blebbing in NB2a
cells (Hagmann et al., 1999). 2,3 butanedione monoxime (BDM) blocks the interaction of
actin with myosin by inhibiting myosin ATPase activity (Ostap, 2002). Urwyler et al.
(2000) showed that BDM suppresses the formation of lamellipodia, but not of blebs in
polymorphonuclear leucocytes. In Walker carcinosarcoma cells, BDM reduced both the
proportion of locomoting and polarized cells (Keller et al., 2002a).
Cells that lost their polarity did not become spherical, but formed protrusions as shown
for colchicine treated cells. Myosin is found predominantly in the cell body and the rear
(in the case of polarized cells) of Walker carcinosarcoma cells (Keller et al., 2002a).
Chelating extracellular Ca2+ did not prevent NB2a cells from blebbing (Hagmann et al.,
1999). Intracellular Ca2+ chelators could not inhibit polarisation or locomotion in NB2a
cells (Hagmann et al., 1999) and Walker carcinosarcoma cells (Von Tscharner Biino et
al., 1997).
Walker carcinosarcoma cells were capable of maintaining cell polarity and motile activity
at Ca2+ level below 18 nM (Von Tscharner Biino et al., 1997), substantially lower than
basal Ca2+ levels in resting polymorphonuclear leukocytes (100 nM), or the requirement
for solating actin filaments (~ µM range, Oster, 1984).
Drug
Colchicine
(10-5M)
Latrunculin A
(10-7M)
Col (10-5M) &
Lat A (10-7M)
EGTA
BAPTA-AM
CI-959
Staurosporine
Calyculin A
Y-27632
BDM
LPA
KT 5926
Jasplakinolide
Action
MT polymerisation
blocker
Actin polymerisation
blocker
Actin and MT
polymerisation block
Extracellular Ca2+
chelator
Intracellular Ca2+
chelator
Intracellular Ca2+
chelator
Kinase inhibitor
Phosphatase
inhibitor
ROCK inhibitor
Actin-myosin
interaction inhibitor
Inducer of Rho A
pathway
MLC kinase inhibitor
Actin disassembly
inhibitor
Type
NB2a
Result
Frontal actin uncoupling: intensive
blebbing, faster locomotion
Frontal cortex disruption: Loss of
polarity, faster locomotion
Frontal actin destruction: loss of
polarity, faster locomotion
No change in locomotion and
polarity
No change in locomotion and
polarity
No change in locomotion and
polarity
Loss of blebbing
NB2a
Intensive blebbing
WCS
Loss of locomotion, polarity and
blebbing
WCS, PMN
Loss of locomotion and polarity
NB2a
Induction of blebs
NB2a
Inhibition of blebs
Fibroblasts
Inhibition of lamellipodia protrusion
WCS
WCS
WCS
NB2a
NB2a
WCS
Table 1.Summary of selected drug experiments on blebbing cells.
10
Introduction
Receptor
Extracellular
Receptor
LPA
DAG Intracellular
RhoA
PI3K
PKC
GTP
GEF
Cdc42
GAP
GDP
RAC1
RhoA
CPI
?
ROCK
PAK
MLCP
MLCK
LIMK
Myosin
(inactive)
MLC
Myosin
(active)
MLC
Actin
Cofilin
Assembly of actomyosin
filaments
Figure 3. Activation pathway of the actomyosin depending contraction
Scheme based upon reviews and publications by Carr et al. (1991), Sells et al. (1997), Klemke et al. (1997),
Schmidt and Hall (1998), Kaibuchi et al. (1999),Wicki and Niggli (1999), Jimenez et al. (2000), Jones (2000),
Keller (2000), Ridley (2001), Diviani et al. (2001), Watanabe et al. (2001), Wicki and Niggli (2001), Riento and
Ridley (2003), Niggli (2003). Abbreviations: CPI=Myosin light chain phosphatase inhibitor, DAG=diacylglycerol,
GAP=GTPase
activating
protein,
GEF=guanine
nucleotide
exchange
factor,
LIMK=LIM
kinase,
LPA=lysophosphatidic acid, MLC=myosin light chain, MLCK=myosin light chain kinase, MLCP=myosin light chain
phosphatase, PAK=p21 activated protein, PI3K=phosphoinositide 3-kinase, PKC=protein kinase C, ROCK=Rho
kinase.
The drug jasplakinolide (Crews et al., 1986) stops actin filament disassembly
immediately in migrating fibroblasts, a tissue cell line, thereby fully inhibiting protrusion
of the lamellipodium. However, the cell body continued to move forward, i.e. towards the
lamellipodium (Cramer, 1999).
The Rho/ROCK, phosphatidylinositol 3-kinase (PI3K) and protein kinase C pathways are
involved in signaling in motile functions (Wicki and Niggli, 1999, Wicki and Niggli, 2001,
11
Introduction
Niggli, 2003). Although these pathways are much more intricate as depicted in Figure 3,
it explains the results of various drug experiments. The motor of bleb formation is driven
by actomyosin depending contraction (and not by actin polymerisation), and is primarily
running via the Rho A/ROCK signalling pathway.
The live and death of a bleb
The viscosity of the hyaline cytoplasm of Amoeba proteus is more than 5 times lower
than of the rest of the cell (Yanai et al., 1999). The viscosity of Swiss mouse 3T3 cells
was estimated to be in the same range (Albrecht-Buehler, 1982). Blebs protrude from
the hyaline cytoplasm within a few seconds, making them among the most rapidly
extending cell protrusions (Boulbitch et al., 2000). Onset is fast and therefore the initial
stage is seen rarely in living cells. The cortex and the plasma membrane disconnect from
each other during bleb formation (Hagmann et al, 1999), comparable to the formation of
a blister. Blebs grow where the actin cortex is weakened (Cunningham et al. 1992, Keller
and Eggli, 1998a, b, Rentsch and Keller, 2000, Merkel et al., 2002): detachment of the
actin cortex initialises the birth of a bleb. The cell membrane peels off from the base of
the bleb, comparable to the formation of a blister. A hydrostatic pressure fuelled stream
is filling the bleb volume and pushing it outward (Bereiter-Hahn, 1985, Strohmeier and
Bereiter-Hahn, 1987). The volume of the bleb increases linearly in time to form a shape
close to a spherical cap. Interruption of bleb protrusion occurs suddenly and is due to the
formation of a new cortical structure lining the bleb membrane (Hagmann, 1999
Boulbitch et al, 2000, Condeelis, 1993, Cunningham, 1995, Grebecki, 1990). Organelles
are never observed in the bleb (Oliver et al., 1995). After recovery of the cortex in the
bleb, actin polymerisation retracts the bleb to restore the initial situation (Keller et al.,
2002b). A major part of the volume of a new bleb stems from older ones. The remainder,
which could not be accounted for might be recruited from the extracellular space
(Albrecht-Buehler, 1982).
12
Introduction
2.2. BIOLOGICAL ELECTRON MICROSCOPY
2.2.1 ELECTRON MICROSCOPY AND RELATED PROBLEMS
At the beginning of the 1930s, after centuries of technical perfection, further
improvement of microscopic resolution was limited by the wavelength of the light source.
Fascinated by the electron wave/particle theory of De Broglie published just a few years
earlier (De Broglie, 1923), Knoll and Ruska were able to build an electromagnetic lens: a
device that could focus electrons as if they were light (Ruska and Knoll, 1931). Shortly
thereafter, they build the first electron microscope (Knoll and Ruska, 1932). The electron
source – a heated tungsten wire – is emitting electrons accelerated by high voltage. The
electromagnetic lenses are focusing the electrons on a specimen. The electron-specimen
interaction results in an image visible on a fluorescent screen or captured on
photographic film emulsion (Watt, 1997).
The column of the electron microscope must be evacuated since the mean free path for
electrons (100 kV) at ambient pressure and temperature is not exceeding the millimetre
range (Wischnitzer, 1980). Water – abundantly present in biological samples – will
evaporate when introduced in a vacuum (10-6 Atm) of an electron microscope at room
temperature (RT), resulting in detrimental shrinkage and subsequent loss of morphology
(Bowers and Maser, 1988). The vacuum system is inherent to the use of electrons and
electromagnetic lenses and therefore cannot be avoided. Removal of the water content is
required in order to observe a sample at RT in an electron microscope. In the classical
way, the sample is treated with chemical fixatives, subsequently dehydrated in a series
of alcohol baths, and embedded in resins (Hayat, 2000). Severe shrinkage, however,
cannot be avoided (Boyde and Maconnachie, 1979). The use of chemical fixatives has
several
more
shortcomings:
selective
fixation2
(Dahl
and
Staehelin,
1989),
swelling/shrinkage of cells and cell compartments (Lee, 1982), destruction of epitopes
with reduction of antigenicity (Monaghan et al., 1998, Skepper, 2000, Rostaing, 2004).
Resin embedding is necessary to section biological samples in slices that are thin enough
(~ 60nm) for electrons to penetrate. Prior to observation in the microscope, a contrastenhancing step is performed by administering heavy metals to the section (Hayat, 1986).
2.2.2 ALTERNATIVES TO CHEMICAL FIXATION
To circumvent the disadvantages of chemical fixatives, alternatives based on freezing
were established soon (Fernández-Morán and Dahl, 1952). Because there is no chemical
reaction with the molecules in the sample, cryofixation (syn. physical fixation,
cryoimmobilisation) has the advantage of being non-selective, faster, and non-
2
The consequence of the selective fixation is the loss of non-linked molecules upon extraction of water.
13
Introduction
destructive towards epitopes (Gilkey and Staehelin, 1986). Once a sample is cryofixed, it
can be cryosectioned at temperatures below -140°C in a cryomicrotome (Dubochet et al.,
1988) or cryodehydration methods such as freeze substitution (Feder and Sidman, 1958)
and freeze drying (Edelmann, 1978) can remove the water. Once the water in the sample
is removed (or replaced by a solvent), warming without severely damaging the sample
can be performed. In the case of cryosections, the still hydrated sections have to be
observed in a cryo-TEM at temperatures below –160°C.
2.2.3 CRYOTECHNIQUES
Ice crystals
The aim of cryofixation techniques is to immobilize a specimen with high spatial
resolution for optimised preservation of morphology and dynamic processes. When
optimally cooled, the sample becomes vitreous, meaning no ice was generated (Echlin,
1992). The vitreous (non-crystalline) state of water can be regarded as liquid water with
an infinite high viscosity (Moor, 1987). Formation of ice – the crystalline state of water –
is damaging for biological structures since only water molecules are incorporated in the
crystal (phase separation) – which leads to segregation of solutes –, and for inducing
morphological changes (Kellenberger, 1987). Whether or not an aqueous solution vitrifies
depends on the ice nucleation frequency, the ice crystal growth rate and the rate of the
heat removal of the system (Angell and Choi, 1986). Ice nucleation is a thermodynamic
process while ice crystal growth is a kinetic process – depending on the conditions in the
surrounding medium (Bachmann and Mayer, 1987). Ice crystal formation is only
occurring within a temperature window (Figure 4, Robards and Sleytr, 1985) bordered by
the melting point (Tm) and by the recrystallisation temperature (Tr). The objective of
cryofixation is to surpass this temperature window by extracting heat faster from the
system than it can be produced by the crystallisation process (Bachmann and Mayer
1987). Several physical parameters influence the outcome of cryofixation:
Solute concentration: Increasing the solute concentrations reduces the ice nucleation
rate and the window of crystallisation and therefore facilitates cryofixation (Gilkey and
Staehelin, 1986).
Cooling rate: Extreme cooling rates are needed to avoid the growth of ice nuclei (Riehle
and Hoechli, 1973).
Sample thickness: Due to the particularly bad heat conductivity of water (the main
constituent in biological material) high cooling rates at the surface do not necessarily
reflect high cooling rates at the centre (Studer et al., 1995, Shimoni and Müller, 1998).
Pressure: The crystallisation window can be trimmed down more due to anomalities in
the behaviour of water under pressure (Sun et al., 2003).
The recrystallisation point for normal biological tissues is still at debate. The value of
185K proposed here is based on the starting point of the freeze substitution (FS) process,
14
Introduction
which yields generally “well frozen” samples: if ice damage occurred, it is too small to be
detected in FS and resin embedded samples (resolution of ~ 3nm).
H2O
Cytoplasm
204.5 MPa
(Tm)
273
251
(Tr)
Temperature (K)
185
130
(Tr)
Figure 4. Ice crystal formation in pure water (H2O) is taking place from the melting point
(273.15K) until the recrystallisation point (130K). Solutes in the cytoplasm and pressure
(optimum at 204.5 MPa) decrease the melting point and increase the recrystallisation
point, hence narrowing the crystallisation window. Source: Robards and Sleytr (1985).
Solute concentration
The presence of solutes affects the dynamics of ice formation by reducing ice nucleation
rate (Angell and Choi, 1986). The crystallisation window (Figure 4) is narrowed down due
to a decrease of the melting point (Tm) and an increase of the recrystallisation point (Tr).
The minimum or ”nose” (Figure 5) corresponds to the least time to form ice nuclei.
Solutes increase the time needed to form ice crystals, allowing vitreous samples at lower
heat removal rates. In extreme cases, cellular water is replaced by a 2.3M sucrose
solution (Tokuyasu samples), which always leads to vitreous water at any cooling rate
(Tokuyasu, 1973).
Cooling rate
The applied cooling rate is the parameter which is the most prone to technical
improvements.
Slow cooling (<1000 K/s)
Ice crystal formation occurs in the extracellular region (highest probability for ice
nucleation due to lower extracellular solute concentration). Since only water molecules
are incorporated in the ice crystal (phase separation; Robards and Sleytr, 1986), the
extracellular (EC) solute concentration rises gradually, eventually leading to osmotic
suction. Accordingly, intracellular water is extracted from the cells and incorporated in
the EC ice crystals. Finally, also the intracellular components reach an enriched solute
concentration, which reduces ice nucleation speed (Figure 5) and causes the sample to
vitrify. Due to the severe dehydration, the morphology of the healthy living state is not
preserved, but ice crystals do not damage the internal cellular apparatus and therefore
the samples are functional on thawing. In this way, biological cells and tissues can be
preserved in frozen conditions.
15
Introduction
1000 – 100,000 K/s
Intermediate cooling results not only in the formation of extracellular ice crystals but also
of intracellular ice. The cooling rate is too slow to extract the heat produced during
crystallization fast enough (Figure 4, Figure 5) hence the window cannot be surpassed
without ice formation (for samples with typical biological solute concentrations). This
causes eventually severe segregation in the biological structure and damage to the
cellular machinery. The morphology is not preserved and the process is killing the cells,
and they are therefore not functional on thawing.
Tm(C2)
Tm(C1)
Tm(H2O)
Log τ (s)
4
0
-4
-8
H2O
C1
C2
Temperature
Figure 5. The effect of solute concentration on the escape time (τ). τ is the time (in
seconds) needed to form an ice nucleus with a probability of 1. Compared to pure water
(H2O), solutes (at concentration C1 and C2) increase the time needed to form an ice
nucleus as well as minimize the temperature window wherein crystallization occurs
(increase of Tm and decrease of Tr). Vitrified samples are achieved by surpassing the
window fast enough to avoid formation of ice crystals. The concentration of C2 >
concentration C1. Redrawn from Angell and Choi, 1985.
~ 100,000 K/s
Sub-optimal cooling rates will not be able to prevent the formation of ice, but might be
fast enough to keep crystal growth to a minimum. Such “well frozen” samples contain
ice, but the traces (solute exclusion, morphological damage) are smaller then the
resolution obtained in resin embedded samples (~ 3nm). After follow up techniques (e.g.
FS, FD) and resin embedding, no absolute conclusion can be drawn about the state of the
water after freezing (vitreous or well frozen). The only absolute method to assign
vitrification to a sample is by producing a diffraction pattern of a frozen hydrated section
in the cryo-TEM at temperatures below –140°C (Dubochet et al., 1988). Well-frozen,
non-vitreous samples are not suitable for frozen hydrated sectioning since they are too
brittle to section (Al-Amoudi et al., 2004a, b).
16
Introduction
> 100,000 K/s
At fast cooling rates, the temperature window is surpassed before ice crystals can form.
The sample vitrifies and the morphology is preserved. Vitreous samples are required in
the case of cryo-TEM (Al-Amoudi et al., 2004a, b). However, upon thawing vitreous
samples, they re-enter the temperature window and intracellular ice crystal formation will
damage the morphology and kill the cells.
Sample thickness
According to Carslaw & Jaeger (1959), the cooling rate is inversely proportional to the
thickness squared (see appendix 1 for formulae). Based on the heat conductivity
properties of water, cooling rates were simulated for slabs with decreasing thickness
(Studer et al., 1995, Figure 6).
133 K
Time in ms
400
193 K
300
213 K
233 K
253 K
273 K
293 K
200
100
0
173 K
50
100
150 200
250
300
40,000 K/s
Infinite K/s
Cooling rate, 1000K/s
153 K
20
10
20,000 K/s
10
10,000 K/s
5
0
5,000 K/s
50
100
150 200
250
300
Sample thickness in µm
30
Time in ms
20
15
10
5
0
10
20
30
153
173
193
213
233
253
273
293
40
K
K
K
K
K
K
K
K
50
Infinite K/s
20
40,000 K/s
10
20,000 K/s
10
10,000 K/s
5,000 K/s
5
0
10
20
30
40
Cooling rate, 1000K/s
133 K
25
50
Sample thickness in µm
Figure 6. The simulation of cooling rates and temperature distributions in 600µm (top)
and 100µm (bottom) thick water slabs (Studer et al, 1995). The Y-axis corresponds to
the slab surface. The X-axis relates to half of the slab thickness (surface to centre). It
takes around 400 ms to cool down a 600 µm thick water slab below Tr (185K).
Furthermore, about 600 K/s is the maximally achievable cooling rate in the centre,
regardless of the cooling rate at the surface. Reduction of slab thickness to 100 µm
considerably enhances the cooling process in the centre: Tr is reached within 25 ms and a
maximum cooling rate at the centre of 15,000 K/s is reached. Source: Studer et al., 1995.
17
Introduction
Due to the low thermal conductivity of water, high cooling rates cannot be achieved in
the centre of a (600 µm) thick slab, even when infinitely high cooling rates are applied at
the surface of the slab. Reducing the sample thickness, rather than technical
improvement is increasing centre cooling rates. In the case of the “bare grid technique”
(Adrian et al., 1984), a sample of 100nm is plunged into liquid ethane (at ∼ -180°C)
thereby reaching theoretical cooling rates of 1010K/s. Biological samples can be vitrified
to a range of 10-20 µm deep at ambient pressure (Sitte et al., 1987).
High pressure
–21,985°C, the melting point of water at 204.5 MPa (Kanno et al, 1975), is the minimum
temperature at which liquid water can exist without ever freezing (maximal decrease of
Tm, Figure 7). Moreover, the region where metastable liquid water can exist (specified by
the interval between Tm and Th – the homogeneous nucleation temperature) is maximal
near 200 MPa (-92°C at 210 MPa, Chaplin, 2005). Therefore, pressure has similar
consequences as solute concentration: it facilitates surpassing the temperature window.
In the light of cryofixation, pressure can be regarded as a physical cryo-protectant.
Owing to this, at about 200 Mpa, 100 times lower cooling rates result in vitrification.
Alternatively, from the perspective of the sample, 10 times thicker samples (see
appendix 1 for formulae) can be vitrified (Sartori et al., 1993a), making it possible to
achieve vitreous biological samples up to 200 µm thick (Studer et al., 1995, Shimoni and
Müller, 1998).
273
0
Tm
I
III
233
213
-40
Th
II
-60
193
173
-20
-80
100
200
Pressure (Mpa)
300
Temperature (°C)
Temperature (K)
253
-100
Figure 7. Part of the phase diagram of water showing the minimum in the melting
temperature (Tm) and the minimum in homogeneous nucleation temperature (Th) near
200 MPa. The shaded area represents the region where metastable liquid water can
occur. The Roman numericals reflect the different types of ice. Redrawn from Kanno at al.,
1975.
18
Introduction
Cryoprotectants
Biologically inactive molecules can enhance the solute concentration and in this way act
as cryo-protectant. High molecular dextran (>40 kda) is an often-used molecule to
facilitate vitrification of the surrounding medium. Dextran is added to the sample shortly
before fixation. 20% dextran samples were found to be vitreous up to 400 µm thick when
cryofixed using high-pressure freezing (Sartori et al., 1993a, b).
Remark
The outcome of cryofixation is a complex process and the values given are merely
guiding rules. Within organs, tissues and even cells a different result is possible after
cryofixation. Therefore, it is almost impossible to predict beforehand if a sample will be
vitreous or not, even if the cooling rate, the sample thickness and the pressure are
optimal.
2.2.4. HIGH PRESSURE FREEZING ARTIFACTS
Although cryomethods are superior over chemical fixation methods in the preservation of
the morphology (Plattner and Bachmann, 1982, Müller, 1988, Von Schack et al., 1993,
Studer et al., 1989, Studer et al. 1992, Studer et al. 1995, Bohrmann and Kellenberger,
2001) as well as in the retention of the antigenicity (Monaghan et al., 1998, Skepper,
2000, Claeys et al., 2004), artifacts can not be avoided. In contrast to chemically induced
artifacts of the classic fixation, artifact induction in high pressure frozen samples is
physical in origin. Discussion of ice crystal artifacts (Escaig, 1982), resin induced
aggregation (Kellenberger, 1987) and ion distribution (Zierold, 1991) are not within the
scope of this manuscript.
Lethal effects of 200 Mpa
During high-pressure fixation, living organisms are submitted to a pressure of 200 MPa
for a period of about 25ms before freezing commences (Studer et al., 2001).
Experiments at 30°C with Staphylococcus aureus revealed a D-value3 of 180 minutes
(Butz et al. 1990), which results in a survival rate of 99.99% after 25ms. Eukaryotic
samples showed an equal insensitivity. An estimated 99.99% of a Saccaromyces
cerevisiae population will survive upon treatment with 200 MPa at 40°C for 25ms (based
upon D value of 10.9 min., derived from Abbildung 1 in Butz and Ludwig, 1991). Survival
rates around 90% for Euglena gracilis algae were published (Riehle and Hoechli, 1973,
Moor, 1978, Moor and Hoechli, 1970) published after treatment with 200 MPa, but for
considerable longer periods (100ms).
High pressure can be used as a technique for sterilization, but many cycles of several
hours (from five hours on) are required (Von Mentrup et al., 1988).
3
see Appendix 2 for definitions and calculations.
19
Introduction
Subcellular level
Although not lethal, high pressure has an effect on the biological structure. MacDonald
(1984) calculated that pressure of 200MPa causes solidification of lipid bilayers from the
liquid-crystalline state to the gel state, resulting in a change in the appearance of the
membranes (Semmler et al., 1998).
High pressure is shown to destroy all structure in vitrified samples of DNA in a
spontaneously formed cholesteric liquid crystal phase (Leforestier et al. 1996), since a
similar sample vitrified with slam freezing (a cryofixation method at ambient pressure)
could retain the structure.
In addition, changes in the microtubule system in Tobacco leaf cells were reported (Ding
et al., 1991, Ding et al., 1992).
20
Introduction
2.3. FAST SAMPLING FOR HIGH PRESSURE FREEZING
Sampling is the first step in a series of procedures, which will inevitable introduce certain
artifacts and thereby deteriorating the original healthy living state of biological material
(Jakstys, 1988).
In the case or viruses and single cells, a situation can be created close to the natural
environment minimizing pre-fixation artifact induction (e.g. the bare grid technique of
Adrian et al., 1984). Tissues, however, require sizing down before fixation. Autolytic
processes commence immediately after excision and the small samples are very prone to
water evaporation (Van Harreveld and Crowell, 1964, Van Harreveld et al., 1965).
When applying chemical fixation, infusion-fixation is the fastest way to fix the material
(Bowers and Maser, 1988). In the case of cryomethods, the sample has to be sized down
before fixation because only small samples can be sufficiently well frozen.
Van Harreveld et al. (1965) suggested 30 seconds between living organ and fixation as
the limit for good preservation of the natural state of mouse neocortex tissue. In
contrast, the rate of most enzymatic reactions and ion diffusion (Na+, K+, Cl- and Ca++)
are many orders of magnitude faster (Bachmann and Mayer, 1987). A diffusion speed of
2 µm per millisecond for the above-mentioned ions was estimated by Zierold (1991).
These calculations were based on diffusion in pure water and the author reasons that
diffusion in biological systems is probably slower.
Biopsy systems require the excision and transfer within 30 seconds, of a sample small
enough to avoid ice crystal growth. Furthermore, it should be easy to handle and the
impact with the target must be kept to a minimum.
21
Introduction
2.4. THE STATE OF WATER AND IONS IN THE CELL
2.4.1 CELL WATER
Ample experimental data reveal that the cytoplasm is not reflecting the state of an
aqueous solution (e.g. Gross, 1987, Clegg, 1988, Negendank, 1988). Water molecules
will orientate themselves as polarized multilayers around charges, a state of water which
behaves differently compared to water in aqueous solutions.
2.4.2 LING’S FIXED CHARGE HYPOTHESIS
Theory
According to Ling (1952, 1962), full ionic dissociation only occurs in dilute solutions of K+
and Na+ salts of monomeric anions (e.g. Cl-). In living cells, K+ and Na+ ions are
associated with charge bearing proteins, which are polymeric, fixed charge systems. The
forces that keep the cations fixed are thermodynamic as well as kinetic in nature. Spatial
fixation of an ion is caused by the overlap of the attracting fields of the ion and the
charged protein. Entropy is considerable reduced due to the confinement of the cation in
a much smaller space, which strengthens the association with the protein. Force
attraction between ions of opposite sign in solutions is opposed by the kinetic energy of
the ions themselves. The number of effective collisions (mostly received from
surrounding water molecules), tearing apart associated pairs is at least halved when one
species is fixed and thus unmoved upon collision (Ling, 2001).
Experimental evidence
For 0.2M sodium isobuteric acid, activity coefficients of 0.9 were found (indicating that
90% of the Na+ is free). However, joining the isobutyric acids in a linear polymer
(polyacrylic acid), thereby fixing the carboxylgroups in space, the activity coefficient of
Na+ dropped to 0.168 (Kern, 1948). Ling (2001) reports the measurement of an eight
times lower diffusion coefficient for K+ (Dk) in living frog sartorius muscle compared to a
0.1M KI solution. However, muscle cells killed with metabolic toxins produce a Dk
comparable to a 0.1M KI solution. In injured cytoplasm, the Dk value lays amid (roughly
3 times lower than the 0.1M KI value). Microelectrode techniques, electroporation and
patch clamp cause injuries in the cell membrane without lethal consequences (Pollack,
2001). Absence of immediate resealing of the membrane is confirmed since the puncture
can be seen by direct microscopic observation (Nickels, 1970, Chang and Reese, 1990).
Sectioned cells survive for use in electrophysiological studies without resealing of the
membrane (Cameron, 1988, Krause et al., 1994). Moreover, substantial penetration of
DNA molecules has been observed 20 minutes after electroporation (Xie et al., 1990,
Klenchin et al., 1991). Thus, pores thousands of times larger than a hydrated ion are
22
Introduction
kept open for long periods without substantial loss of the ion balance, reflecting the view
that most of the ions are not fully dissolved in the cellular water and that membranes do
not appear to be essential for the retention of ions and proteins (Hazlewood and
Kellermayer, 1988).
Counter anions
Since chloride is not abundant enough in the cell cytoplasm to account as anionic
counterpart for all intracellular cations, it is reasonable to assume that the anion is
organic in nature. Considering red blood cells, haemoglobin (97% of the protein content)
inevitably provides cations adsorption sites. Titration of a haemoglobin solution with
NaOH showed a one-on-one correlation between adsorbed alkali-ions and the replaced
Na+ ions (Ling and Zhang, 1984).
Titration of a muscle segment with acid will replace the alkali ions for H+ ions. The pKa of
about 3.9 can be calculated from the titration curve (Figure 8, left) points in the direction
of involvement of β and γ carboxyl groups (pKa = 3.95) of aspartic acid and glutamic
acid.
120
Probability
[Na+]in (mM)
100
80
60
40
K+
Na+
20
0
-O0
1
2
3
4
5
pH
6
7
8
9 10
2
4
6
8
Distance (Å)
Figure 8. The graph on the left shows the titration curve of adsorbed alkali-metal ions in
a muscle segment upon variation of the pH. Arrow: the pH at which half of the absorbed
alkali ions are replaced (pKa). Source: Ling and Ochsenfeld, 1991. The graph on the right shows
a Boltzmann distribution curve reflecting the probability of finding a positive ion at any
given distance from the centre of a fixed oxyacid (-O-) atom (white, Ø 2.8Å). Note the
difference in diameter between the hydrated Na+ ion (dark grey, Ø 5.6Å) and K+ ion
(light grey, Ø 4.0Å) is sufficient to attain the effect of a majority of anionic sites being
associated with K+. Black dots represent the centre of the ion. Source: Ling, 1952.
Treatment with carbodiimide, a carboxyl selective reagent, leads to the loss of all the β
and γ carboxyl groups and their negative charges. Titration of carbodiimide treated
muscle cells revealed that alkali ions exist as free ions and not as adsorbed ions (Ling
and Ochsenfeld, 1991). In fact, carbodiimide leads to the loss of all carboxyl groups and
not only the β and γ carboxyl groups. But given the large amounts of anorganic cations (∼
80mM in muscle cells), only the β and γ carboxyl groups of aspartic acid and glutamic
23
Introduction
acid are numerous enough to provide a uniform population of adsorption sites (Ling and
Ochsenfeld, 1966).
Nature of the intracellular cation
Na+ and K+ are the most abundant alkali-ions involved in living cells, with K+
predominantly accumulating intracellularly, while Na+ is the most abundant alkali ion in
the extracellular space. Ling (1952) showed that a metabolic pump (K+/Na+ ATPase) is
not the only explanation for this distribution pattern. Based on the size of the hydrated
ions (Ø K+hyd = 4.0Å, Ø Na+hyd = 5.6Å), potassium can infiltrate more deeply in the fields
of the coulombic forces exerted by the negative charges of the above-described β and γ
carboxyl groups (Figure 8, right). Therefore, Na+ can be excluded from the cytoplasm in
favour for K+ (Ling and Zhang, 1983a,b).
57% of the proteins in frog muscle cells are myosin, and 17.5% of the myosin protein
carry free β and γ carboxyl groups. Furthermore it is estimated that 66% of all β and γ
carboxyl groups are found in myosin (Ling and Ochsenfeld, 1966), and a major part of
the absorbed K+ ions must be found associated with myosin. Autoradiographs of
cryofixed and FD frog sartorius muscle published by Edelmann (1988) show a K+
accumulation in the A-band, where all myosin is located.
2.4.3 SALT LINKAGE
Selective adsorption of cations is not regarded as involving the formation of rigid bonds.
The ions are hydrated and dissociated, with a statistical probability that mainly K+ ions
will occupy fixed anionic sites (Ling, 1992).
Apart from anorganic cations, also organic cations can engage in bonds with β and γ
carboxyl groups. Intracellular proteins bearing positively charged ε aminogroups (lysine)
or guanidyl groups (arginine) are the main candidates (Ling, 2001). Chemical energy,
supplied by ATP, can be used to exchange a ε-amino or guanidyl residue for a K+ ion.
(Ling, 1992, 2001).
24
Results
3. Results
3.1. PUBLISHED RESULTS
A rapid microbiopsy system to improve the preservation of biological samples prior to
high pressure freezing4
see attached reprint
4
Published as Vanhecke, D., Graber, W., Herrmann, G., Al-Amoudi, A., Eggli, P., D. Studer, D. (2003) A rapid
microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing. Journal of
microscopy 212(1): 3-12. A technical description concerning the use of our microbiopsy needle was prepared as
Vanhecke, D., Eggli, P., Graber, W., Studer, D. (2005) A new microbiopsy system enables rapid preparation of
tissue for high pressure freezing In: Cell imaging techniques (Taatjes, D, ed), Humana press, NY, USA, In
press.
25
Results
3.2. MANUSCRIPT IN PREPARATION: HITHERTO NOT
DESCRIBED INCLUSIONS IN THE FRONT OF
WALKER
CARCINOSARCOMA CELLS MAY PLAY A ROLE IN
CELLULAR MOTILITY
Introduction
In the next pages I describe a cytoplasmic inclusion named pseudovacuole (PV), which is
found at the front of Walker carcinosarcoma cells. The pseudovacuole might be involved
in the process of cell motility, especially in the formation of blebs.
Since the first cellular locomotion model was proposed by Mast (1926), numerous
attempts were made to explain motility at the sub-cellular level. Most models proposed
rely either on actin treadmilling (Taylor et al., 1980, Wang, 1985, Zhu and Skalak, 1988,
Sheetz et al., 1992, Bretscher, 1996, Svitkina et al., 1997, Cramer, 1999, Small et al.,
1999, Waterman-Storer and Salmon, 1999, Yosida and Inouye, 2001, Paku et al., 2003,
Rafelski and Theriot, 2004) or on pressure (Dipasquale, 1975, Bereiter-Hahn et al. 1981,
Bereiter-Hahn and Strohmeier, 1987, Oster and Perelson, 1987, Dembo, 1989, Grebecki,
1994) as means for propulsive force. Moreover, it was suggested that the diverse motile
functions of cells (e.g. locomotion, blebbing, phagocytosis) might be driven by more than
one underlying mechanism (Boulbitch et al., 2000, Keller et al., 2002).
Grebecka and Grebecki (1981) opened the pseudopodium tips of the free-living Amoeba
proteus and observed that the cytoplasm was squeezed out through the breach into the
external medium. Yanai and colleagues (1996) carried out intracellular hydrostatic
pressure measurements in the same species and recorded a six-time internal pressure
increase (up to more than 30 cmH2O) upon pseudopod formation. Applying external
suction pressure of 30 cmH2O caused spreading in fibroblasts (Thoumine and Cardoso,
1999). Furthermore, fibroblast cytoplasmic viscosity was significantly higher in nonspreading fibroblasts, a feature that could not be undone by cytochalasin (Thoumine and
Cardoso, 1999). In the case of Amoeba proteus, Yanai et al. (1999) could show a
significantly lower viscosity and an almost complete loss of stiffness in the developing
pseudopod compared to the body and trailing region.
Using a micropipette suction technique, evidence that hydrostatic pressure also plays a
role in the motility of Walker carcinosarcoma cells was gathered by Schütz and Keller
(1998). They showed that cells undergoing suction pressure induced deformations
develop contractile responses in the form of constriction rings and can form protrusions
upon relaxation. Further suction pressure experiments revealed that the plasma
26
Results
membrane can be uncoupled from the underlying cortex (Rentsch and Keller 2000) to
form blebs. In Dictyostelium discoideum, twice as much suction pressure with a
micropipette is required to aspirate the trailing edge than the leading edge, but in
knockouts mutants for talin, a membrane associated protein important for the coupling
between membranes and the cortex, no such topographical differences were observed
(Merkel et al., 2000). During this experiment, GFP labelled actin showed the
disconnection of the actin cortex from the membrane.
Hypertonic media decreased motility in corneal epithelian cells (Dipasquale, 1975).
Similar experiments carried out later on with Walker carcinosarcoma cells showed that
both locomotion and motility (blebbing) are suppressed within 10 seconds upon applying
200mM sorbitol and polarity and pre-existing protrusions disappeared after 5-10 minutes
(Fedier and Keller, 1997). Aortic smooth muscle cell migration showed a dose-depending
inhibition of motility towards increasing sucrose concentration in the surrounding medium
(Schousboe, 2003).
Colchicine (10-5M) treatment of Walker carcinosarcoma cells inhibits microtubuli
polymerisation and is indirectly causing the uncoupling of the frontal cortex from the
plasma membrane (Keller, 2000). Cells treated this way showed heavy blebbing in the
front of the cell. It was proposed that vesicle formation in cytochalasin treated
fibroblasts, and the onset of protrusive processes in Amoeba are due to the separation of
actin from the cell membrane (Thoumine and Cardoso, 1999, Taylor et al., 1980).
Combined treatment of colchicine (10-5M) and Latrunculin A (10-9M) resulted in a
complete frontal actin deficiency of Walker carcinosarcoma cells, leading to a loss of
cellular
polarity
and,
contradictory,
a
higher
locomotive
speed
(Keller,
2000).
Polymorphonuclear leucocytes treated with 2,3-Butanedione monoxime (BDM), a myosin
inhibitor, resulted in the inhibition of shape changes and, at a somewhat higher
concentration, locomotion (Urwyler et al., 2000).
Walker carcinosarcoma cells were shown to establish cellular polarity as well as
locomotion at very low intracellular Ca++ concentrations (<18nM) as well as in the
absence of extracellular Ca++ (Von Tscharner Biino et al., 1997). These Ca++ levels are
well below the Ca++ concentrations in locomoting Polymorphonuclear leucocytes (100 nM,
Gustafson and Magnusson, 1992) and the concentrations needed for solating actin
filaments (micromolar range, Oster, 1984). Initial bleb formation protruding at higher
speed than actin filament-growing rate can achieve (Keller and Bebie, 1996) indicates
the use of a hydrostatic pressure driven motility system rather than a frontal actin
polymerisation driven system.
Also other blebbing cell lines were shown to lack actin polymerisation upon bleb
formation. Hagmann et al. (1999) showed in NB2a rat neuroblastoma cells that the actin
cortex is absent in newly formed, protruding blebs, but present in older, retracting blebs.
27
Results
The cortex disconnects from the phospholipid bilayer in Dictyostelium cells (Boulbitch,
2000) and Walker carcinosarcoma cells (Rentsch and Keller, 2000) and this disconnection
is needed for the onset of blebbing. The remnants of the actin cortex of a newly formed
bleb were found at the base of the bleb and were called “restriction rings” (Fedier et al.
1999). Such a hydrostatic pressure system could be driven either by actomyosin
contraction, by osmotic pressure, or by both.
Evidence is available that full ionic dissociation of K+ and Na+ salts only occurs in dilute
solutions (Ling, 1952). In living cells, K+ (and to a much lesser extent Na+) associates
with charge-bearing proteins resulting in polymeric, fixed charge systems (Ling’s fixed
charge hypothesis, Ling, 1952, 1960). According to this hypothesis, negatively charged β
and γ carboxyl groups (of respectively aspartic acid and glutamic acid) in native proteins
are largely engaged in salt-linkages with cations (K+) or with so called fixed cathions:
positively charged ε-amino groups and guanidyl groups (of lysine and arginine residues
respectively). Using ATP as energy source, β and γ carboxyl groups can be set free from
their fixed cations and made available for adsorbing free K+ ions (Ling, 2001: chapter
10).
Here I introduce a new cytoplasmic entity that might – in my belief – be involved in
Walker carcinosarcoma motile functions. The inclusions found at the front of the cells,
termed pseudovacuole, are described using light microscopic, fluorescent and electron
microscopic techniques. The concentrated proteins in the pseudovacuole may build up
pressure according to Ling’s fixed charge hypothesis (LFCH).
Material and methods
Cell culture
Walker carcinosarcoma 256 subline 1 is a mammalian cell line (Sroka et al., 2002).
Walker carcinosarcoma cells were grown in suspension at 37°C (5% CO2 added to the
atmospheric settings) in RPMI1640 medium (Invitrogen, Basel, Switzerland) with a
supplement of 10% fetal calf serum (FCS, Invitrogen, Basel, Switzerland). Cellular
density was kept at 500.000 to 1.000.000 cells/ml. Culture flasks were changed twice
per week to prevent the transformation of the culture to subline 2 (adherent cells
described by Sroka et al., 2002).
Differential interference contrast
Differential interference contrast observations were carried out on a Olympus Vanox
microscope (Olympus, Hamburg, Germany). Glass slides and cover glasses were
preheated and kept at 37°C using a homemade heating plate fitting the Vanox
observation stage. For long-term observations (> 10 minutes), the cover glass was
28
Results
sealed airtight by means of paraffin. Image recording was done using a Canon 10D
digital camera (Canon, Tokyo, Japan).
Sample preparation for light scanning microscopy
10 µM (i.e. 10 microliter of a 10mM stock solution in DMSO diluted in 10 ml culture)
Carboxyfluorescein diacetate succinimidyl ester (CFDA SE cell lysine tracer kit, V-12883
obtained from Invitrogen, Basel, Switzerland, Ex/Em 492/517nm) is a membrane
permeable amine marker, labeling all lysine (residues and incorporated) in the cytoplasm
and the cell nucleus. CFDA SE was applied at 37°C to the Walker carcinosarcoma cells
and was incubated for 10 minutes at 37°C. Subsequently, the cells were centrifuged (5
minutes at 1200 rotors/min, 196mm radius) and rediluted in fresh preheated RPMI
1640+10%FCS medium and allowed to recover for 1h at 37°C. The lipid marker DiIC18
(part of the Vybrant Multicolor Cell-Labeling Kit, V22889 obtained from Invitrogen, Basel,
Switzerland, Ex/Em 549/565nm) is a cell permeable dye which displays red fluorescence
after being incorporated in a hydrophobic region in the cytoplasm or the cell nucleus. DiI
was applied for 10 minutes in a 1µM concentration (10 µl of a 10 mM stock solution
added to 10 ml of cell suspension) at 37°C and washed out as described for the CFDA SE
tracer. SYTO17 was obtained as a part of a SYTO Red Fluorescent Nucleic Acid Stain
Sampler Kit (S11340, Invitrogen, Basel, Switzerland, Ex/Em 621/634nm). SYTO17, a cell
permeant nucleic acid stain (DNA as well as RNA), was applied at 1 µM concentration for
30 minutes at 37°C. After incubation, the cells were centrifuged (5 minutes at 1200
rotors/min, 196mm radius) and diluted in fresh, 37°C preheated RPMI 1640 medium
(+10% FCS). 20 µl drops of labelled cells were observed between two round airtight
sealed 42 mm diameter slides (LaCon, Karlsfeld, Germany), separated by a 0.6mm thick
spacer. An approximately 40 times larger atmospheric volume, present as well between
the slides, prevented introduction of hypoxic artifacts. Observation was carried out in a
Zeiss META 510 light scanning microscope (Zeiss, Oberkochen, Germany), at controlled
temperature parameters using a LaCon heat stage (LaCon, Karlsfeld, Germany). Digital
Image acquisition was done using the Zeiss LSM 510 meta imaging software.
Cell count analysis
10 ml of a dense Walker carcinosarcoma cell culture (1.106 cells/ml) was labelled with
CFDA SE and 20 µl was inserted between two LaCon 42 mm slides as described before.
Positioning was done randomly, without visual selection. Z-axis depth location was
achieved using the “search and focus” function of the Zeiss LSM software and recording
was using a 40x apochromatic oil objective with an extra 2x digital magnification by the
Zeiss LSM 510 meta imaging software.
29
Results
Hypertonic media experiments
20g sucrose (Merck, Darmstadt, Germany) was diluted in 80g RPMI1640 medium (10%
FCS included) resulting in 20% sucrose solution (0.58M). 2.5 ml and 5 ml of this solution
was mixed with respectively 7.5 ml and 5.0 ml cell suspension in RPMI1640 medium
(with 10% FCS) in order to obtain Walker carcinosarcoma cells in RPMI1640 medium with
increased hypertonicity (0.15M and 0.29M).
Sample preparation for high pressure freezing
50 ml of a Walker carcinosarcoma cell suspension with cell densities between 800.000
and 1.000.000 cells per millilitre were centrifuged (5 minutes at 1200 rotors/minutes,
196mm radius) in RPMI1640 medium+10%FCS one day before the start of the
experiment. At any time, the cells were kept at 37°C by means of a warm water bath
(Memmert, Schwabach, Germany) or a home-made heating plate. 1 ml of cell suspension
was centrifuged for 30 seconds at 2000 RPM in a deskspin (Biofuge Pico, Heraeus,
Hanau, Germany). Freeze fracture carriers (Leica, Vienna, Austria), loaded on the Leica
microbiopsy transfer system (Leica, Vienna, Austria) were used as specimen holders. The
central cavity of the carrier is filled with pellet and immediately sealed in the Leica
EMPACT specimen holder (Leica, Vienna, Austria) and high pressure frozen as described
earlier (Studer et al. 2001, Vanhecke et al., 2003).
Freeze substitution, resin embedding, ultra thin sectioning and TEM
Freeze substitution in dehydrated aceton containing 2% osmium tetroxide was carried
out in the Eppendorf system of a Leica AFS (Leica, Vienna, Austria) as described by
Studer et al. (2001). The osmium tetroxide was obtained from Oxkem ltd (Reading,
United Kingdom) and acetone from Merck (Darmstadt, Germany). Acetone was stored
over calciumchloride in order to be free of water.
Epon embedding was done over 4 steps at increasing concentrations: 1:2 (overnight),
1:1 (overnight) and 2:1 (overnight) in waterfree aceton and finally pure Epon
(overnight). Previous to the dilution in acetone, 1.5% N-benzyl dimethyl amine (BMDA,
Fluka, Buchs, Switzerland) was added to the Epon as a polymerisation initiator. The
copper specimen holders were removed during the transfer of the samples to the fresh
Epon (+1.5% BMDA) in the polymerisation mold. Polymerisation was carried out by heat
at 60°C for 72 hours.
Ultra thin sections (50nm) were obtained on a Leica Ultracut S (Leica, Vienna, Austria)
equipped with a 35° diamond knive (Diatome, Nidau, Switzerland) and collected on 200
mesh copper grids (Electron microscopy sciences, Hatfield, PA, USA) covered with
parlodium and a carbon film. The sections were observed in a Philips EM300 transmission
electron microscope (Philips, Eindhoven, The Netherlands) at 60 kV and recordings (1
30
Results
second exposure time) were made on Kodak electron S0-163 image negatives and
scanned using a HP scanjet 7400c dia/negative scanner at 600 dpi.
LM, LSM and EM correlation pictures
Walker carcinosarcoma cells were labeled with CFDA SE (Ex/Em 492/517nm, as
described before) and high pressure frozen immediately after washing out the remnants
of the fluorescent label. After FS (with OsO4) and Epon embedding, one semi-thin section
(toluidine blue stained) and 2-3 ultra-thin sections (70 nm) were made and correlated
with LSM recordings, obtained by scanning the block face in the LSM according to Biel et
al. (2003). Immersion oil, required to make the LSM scans could not be removed
sufficiently to allow clear sectioning. Therefore, LSM scans were made after ultra-thin
sectioning, and a slight shift (2-3 µm) between the LSM and the TEM micrographs could
not be avoided.
Digital image handling
Brightness and contrast of digital images were enhanced using Adobe Photoshop version
6.0.
31
Results
Results
Untreated Walker carcinosarcoma cells observed in a differential interference contrast
(DIC) microscope display a large intracellular inclusion in the frontal part of the cell,
predominantly found in the direct vicinity of blebs, which appears to be a vacuole.
Transmission electron microscopy and fluorescent lipophylic markers failed to show the
existence of a biological membrane surrounding the inclusion and therefore it is not an
organelle. The term “pseudovacuole” (PV) was chosen for its resemblance with a vacuole
and to emphasize the difference from it.
Even for the trained eye pseudovacuoles of Walker carcinosarcoma cells can be difficult
to recognize under brightfield optics. To unequivocally attribute a pseudovacuole in the
light microscope, DIC optics and even better fluorescence labeling (respectively revealing
its borders and its content) are required.
DIC reveals pseudovacuoles as round, ovoid or sausage-like structures with a
homogeneous content sharply bordered from the rest of the cytoplasm (Figure 9 and
Figure 10). The coarse-grained cytoplasm of the cell body surrounding the nucleus
abruptly changes near the front in a non-granular, hyaline cytoplasm, where blebbing
occurs (Figure 9B and Figure 10). The pseudovacuole is found in the frontal, hyaline part
of the cell. During blebbing, the coarse-grained cytoplasm in the cell body stays rigid.
Intracellular movement of the pseudovacuole could be shown using time-lapse video
(Figure
10).
Movement
was
infrequent
and
slow
compared
to
blebbing.
The
pseudovacuole remains as a single entity or, much less frequently observed, as two or
more inclusions.
Pseudovacuoles were present in the cell for the time of the experiment (>1 hour).
Pulsating or contractile behaviour, similar to the contractile vacuole activity in e.g.
Dictyostelium (Gabriel et al., 1999), was not observed. As summarized in Figure 11, the
majority of the cells was found polarized (54.6%) and was containing a pseudovacuole
(62.1%). Pseudovacuoles were found in polarized as well as spherical cells. Observation
of 150 blebbing cells revealed a pseudovacuole in more than 90% of the cases in the
immediate vicinity of the blebs (Figure 12). Moreover, blebbing behavior seized when the
inclusion moved and blebbing restarted at the new location of the pseudovacuole (Figure
10). Suboptimal treatment of the cells (4h at room temperature) resulted in a much
lower occurrence of the pseudovacuoles: in only 14.5% of the cells a pseudovacuole
could be observed, compared to 62.1% for the control cells. This suggest a breakdown of
the pseudovacuole over sustained suboptimal (room temperature) environments, and
hence an active actor in the biology of the cells and not an artifact.
Hypertonic media influenced the occurrence of blebbing. In control populations, more
than 90% of the cells established blebs, which is in accordance with Keller (2000). 5%
32
Results
sucrose resulted in a decrease of blebbing occurrence to 66%. Increased sucrose
concentrations (10%) further decreased the blebbing rate to 28% (Figure 13).
Prolonged observation periods were avoided to prevent artifactual processes, but
observation periods as long as two hours did not reveal any noticeable change in
pseudovacuolar volume. Fusing, dividing, shrinking or growing of the pseudovacuole was
not observed. The fate of pseudovacuoles during cell division was not investigated
(roughly 0.5-1% of the cells are in the M phase).
Basic pseudovacuolar content analysis was carried out using three chosen fluorescent
labels (Figure 14): a nucleic acid dye (SYTO17), a lipid staining hydrophobic dye (DiI)
and an amine reactive dye (lysine tracker CFDA SE, protein staining).
The nucleic acid dye (SYTO17) shows the presence of all nucleic acid content in the
nucleus (DNA and RNA) as well as in the cytoplasm (RNA, tRNA, ribosomes). The dye
was found throughout the entire cytoplasm, but pseudovacuoles turned out to be lacking
nucleic acid content, as the fluorescent signal in the pseudovacuoles was close to the
background level (Figure 14B). Higher levels of the nucleic acid dye were found in a
distinct region in the nucleus (the nucleolus), surrounding the nucleus (showing the
nucleic acid rich rough endoplasmatic reticulum system) and at well-defined spots in the
cytoplasm (probably marking mitochondrial DNA).
The lipid label (DiI) disproved a hydrophobic content of the pseudovacuole, the
pseudovacuoles being one of the least hydrophobic regions in the cell (Figure 14C).
Furthermore,
a
hydrophobic
layer
surrounding
the
pseudovacuole,
indicating
a
pseudovacuolar membrane, was not found (Figure 14C inset). Strong fluorescence was
seen in the nucleolus and in membrane rich systems such as the endoplasmatic reticulum
surrounding the nucleus and the cristae of the mitochondria (Figure 14C).
Lysine, a basic, ⊕-charged amino acid, proved to be more abundant in the pseudovacuole
compared to any other part of the cell (Figure 14D). The lysine tracker CFDA SE shows
that the pseudovacuole is a highly amine-rich region, implicating a protein filled entity
and an abundance of lysine. Together with the practical absence of the hydrophobic
marker DiI, this observation points in the direction of a possible function involving waterrelated processes (e.g. ion balance, osmolarity) for the pseudovacuole. Fluorescent signal
of the lysine label CFDA SE was absent in small spherical bodies, mostly grouped
together (Figure 14D and Figure 15C). No overlap was found between these bodies and
mitochondria which positively labeled with the hydrophobic dye and the nucleic acid dye.
The CFDA SE negative bodies are probably endosomes.
45 cells (10.9% of all cells) appeared to be burst (Figure 14D). These cells often (42
cells, 93.3%) held a pseudovacuole in the erupted region of the cell. Burst cells were not
included in the previously described calculations.
Transmission electron microscopy (TEM) of chemical fixed cells revealed the occurrence
of large electron transparent inclusions in the Walker carcinosarcoma cells (Figure 16).
33
Results
However,
chemical
fixatives
were
not
able
to
preserve
the
content
of
the
pseudovacuoles. Improved preservation of the morphology was obtained in a large range
of biological tissues using cryomethods (McDonald and Morphew, 1993; Royer and
Kinnamon, 1996; Studer et al., 1992 ;Von Schack et al., 1993; Bohrmann and
Kellenberger, 2001; Eggli and Graber, 1994; Studer et al., 1995; Hunziker et al., 1997;
Hernandez-Verdun et al., 1991; Kiss et al., 1990). To exclude artifact formation prior to
fixation, sampling was performed as fast as possible: It took less than 1 minute to
transfer the cells from a flask with cell suspension at 37°C until the cells are high
pressure frozen in the Leica EMPACT (Studer et al., 2001, Vanhecke et al., 2003).
Furthermore all tools were kept at 37°C throughout the sampling prior to high pressure
freezing.
Observations of HPF cryofixed Walker carcinosarcoma cells confirmed the presence of
inclusions corresponding to the pseudovacuoles in size, shape and location. Hence, the
inclusions are not an artifact of chemical fixation or of high pressure freezing. In contrast
to chemically fixed Walker carcinosarcoma cells, inclusions in high pressure frozen
samples did not appear to be empty (Figure 17). Loosely ordered structures, absent in
chemical fixed specimen, made up the inclusion’s content (Figure 17B, 18E). No
organelles or ribosomes were observed in the inclusions, and furthermore, a surrounding
membrane could never be revealed (Figure 17B and Figure 18E) confirming the DiI
labeling results.
The inclusions observed in TEM coincide in location, size and form with the
pseudovacuoles of DIC observations and the fluorescent signal of CFDA SE labeled cells.
Moreover, optical sectioning of CFDA SE labeled HPF frozen Epon embedded cells allowed
comparison of LSM data and TEM data, and the strong fluorescence of the pseudovacuole
co-localized with the inclusion observed in the TEM (Figure 18A-C). The inclusions seen in
figure 18D co-localize with the fluorescent stain of the pseudovacuole (figure 18F).
Disconnection of the cortex from the cellular membrane, as the onset of blebbing, could
be observed on several occasions (Figure 19).
34
Results
Discussion
Pseudovacuoles are extremely difficult to observe in brightfield optics. Differential
interference enhanced contrast facilitates the observation considerably, but nevertheless
observation and assessment remains challenging.
The obscurity of the pseudovacuole is further enhanced by the fast dynamical processes
in the front of the cell (bleb formation), distracting attention from pseudovacuoles, which
display much slower dynamics. Additionally, Z-axis aligned protrusions can easily be
mistaken for pseudovacuoles.
Breakdown of pseudovacuoles is seen indirectly in less favorable circumstances (room
temperature treatments), which implies a biological relevance for the pseudovacuoles
and not a passive stockpile artifact.
Evidence is available in favor for the theory that motility in Walker carcinosarcoma cells
is at least partially depending on intracellular pressure (Boulbitch et al. 2000, Rentsch
and Keller, 2000, Keller et al., 2002; Fedier and Keller, 1997; Sroka et al., 2002). As
observed in burst Walker carcinosarcoma cells, outward pushed cytoplasm reveals the
high intracellular pressure (in agreement with the Amoeba proteus observation of
Grebecka and Grebecki, 1981). The disruption is most probably caused by experimental
handlings: centrifugation, pipetting, repeatedly cooling down to room temperature and
warming up to 37°C, or as an effect of aldehyde fixation. Introduction of a 0.6 mm
spacer between the slides prevents mechanical pressure generated by the slides on the
cells during observation. Since the outburst is pressure driven, the observation that the
pseudovacuole is in almost all cases enclosed by the outburst is suggesting that the
pseudovacuole is in or near the epicenter of a frontally directed pressure vector.
Higher locomotive speeds were achieved when the frontal actin cortex was destroyed by
drug treatments (Keller, 2000). This drug treatment is not detrimental for locomotion but
devastating for motile functions, which shows the uncoupling between these processes.
Only myosin blocking agents could stop both blebbing and locomotion (Urwyler et al.,
2000). The idea that diverse motile functions such as blebbing, lamellipodia formation,
phagocytosis and locomotion are regulated by one single mechanism is difficult to
maintain.
In my vision, bleb formation is a pressure induced motile cell function. Experiments done
by Keller (Keller, 2000) revealed that actomyosin contraction in the rear and the cell
body is vital for locomotion as well as motility. The intracellular pressure (Pintracellular) can
be written as the sum of the intracellular hydrostatic pressure (Phydrostatic) and the
intracellular osmotic pressure (πintracellular)
Pintracellular = Phydrostatic + πintracellular
35
Results
According to Pascal’s principle Pintracellular is transmitted undiminished over the entire cell,
and every point of the cell membrane perceives the same pressure.
In the first step of blebbing, actomyosin contraction increases the intracellular
hydrostatic pressure in the cell (Pcontraction)
Phydrostatic = Phydrostatic + Pcontraction
Next, local disassembly of the actin cortex creates a weak point, resulting first in a
breach in the cortex and subsequently in the formation of a bleb. Cortex disconnection
and bleb formation was experimentally shown by Rentsch and Keller (1999) and
Hagmann et al. (1999). Retraction of the bleb is accomplished by actomyosin relaxation
(Pcontraction ≈ 0) and actin assembly in the bleb resulting in cortex resolvement.
Additionally, organelles and ribosomes were not found in the front or in the bleb of
Walker carcinosarcoma cells, nor were they present in the better studied frontal hyaline
cap and pseudopods of Amoeba cells. While restriction rings, layers of actin physically
separating the hyaline front of the cell from the ribosome and organelle rich, coarsegrained cytoplasm in the cell body, might be used to explain this, this is incorrect to my
belief. Since frontal cytoplasm and cytoplasm in the cell body have different viscoelastic
properties (Yanai et al., 1999), during cytoplasmic flow, a gel to sol transition should
take place. This is not probable given the sharp delineation observed between frontal and
cell body cytoplasm. Moreover, the question why organelles (mitochondria, vesicles, …)
are always absent in the bleb would still remain. In the next paragraphs, I propose a
system, which is compatible with all previous data and could explain the blebbing in a
much less complex way.
Pseudovacuoles are present in most Walker carcinosarcoma cells, and always in the
hyaline cytoplasm at the front of the cell, nearly always in the vicinity of blebs. This
suggests that pseudovacuoles might be involved in blebbing motility. I propose that the
pseudovacuole acts as an osmotic regulator, aiding the actomyosin motor to build up
pressure and responsible for attracting the content of the bleb.
According to Ling’s fixed charge hypothesis (LFCH; Ling, 1952), negatively charged β and
γ carboxyl groups of proteins are engaged in salt-linkages with fixed cations like lysine
and arginine side-chains. According to Ling (1952, 2001), ATP occupies key controlling
cardinal sites, which results β and γ carboxyl groups made available for adsorbing
hydrated alkali ions (K+). Accordingly, positive ions are bound or released in a controlled
manner and are thus influencing the internal osmoticity, and therefore, indirectly
influencing the influx of water. Lysine residues were found at high concentrations in the
pseudovacuoles of Walker carcinosarcoma cells.
36
Results
Seeing the pseudovacuole in the light of an osmotic regulator acting according to the
LFCH answers most questions adequately. On one hand, the pseudovacuole helps
increasing the intracellular pressure, since:
π = πinitial intracellular + πpseudovacuole
With πinitial
intracellular
the initial intracellular osmotic pressure and πpseudovacuole the osmotic
pressure induced by releasing positive ions in a controlled way by the pseudovacuole.
Combined with actomyosin contraction the intracellular pressure can be written as:
Pintracellular = Pinitial hyd + Pcontraction + πinitial intracellular + πpseudovacuole
On the other hand, because of intracellular osmoticity increase, an influx of extracellular
water near the pseudovacuole takes place, resulting in sol-like cytoplasm at the front of
the cell. This cytoplasm will fuel the forming bleb. The consequences of the idea are
profound: first, the coarse-grained cytoplasm of the cell body does not take part in the
blebbing of Walker cells, except for the passive transduction of the pressure, hence does
not need a gel-sol transformation. Second, the cell body stays rigid during blebbing,
which is consistent with every observation made on blebbing Walker carcinosarcoma cell.
Third, an osmotic regulator acting in this way must be freely accessible by ions. A
surrounding membrane would be restricting the accessibility and therefore the function of
the osmotic regulator. Membranes were not found around the pseudovacuoles, which is
consistent with its putative function as an osmotic regulator.
37
Results
Figures
Figure 9. Differential interference contrast pictures of untreated living Walker
carcinosarcoma cells at 37°C. Pseudovacuoles are visible as large, smooth inclusions,
deprived of any typical cytoplasmic elements (e.g. mitochondria and vesicles). B=Bleb,
Gc=Granular cytoplasm, Hc=Hyaline cytoplasm, N=Nucleus, P=Pseudovacuole,
U=Uropod.
38
Results
Figure 10. A time lapse recording of an untreated, living Walker carcinosarcoma cell, kept
at 37°C. 5 seconds time interval has passed between two subsequent pictures. The series
reveals a pseudovacuole in the longitudinal axis of a vertically aligned cell, with bleb
formation in the immediate vicinity of the pseudovacuole (0’’). Upon movement of the
pseudovacuole, blebbing ceases at the original spot but keeps proceeding around the
pseudovacuole (e.g. bleb formation at 25’’ and 50’’). B=Bleb, Gc=Granular cytoplasm,
Hc=Hyaline cytoplasm Nb=New bleb, P=Pseudovacuole, N=Nucleus. The uropod cannot
be seen in this focal plane.
39
Results
60
100
90
50
80
70
40
60
30
50
40
20
30
20
10
10
0
0
Total
Spherical
Polarized
Figure 11: Occurrence of pseudovacuoles and morphology of Walker carcinosarcoma
cells. █ cell counts, █ pseudovacuole counts. Polarized cells were more abundant than
spherical cells (54.6% to 45.4%). Pseudovacuoles were found in the majority of the cells
(62.1%). Pseudovacuoles occurred in spherical (54.8%) and polarized cells (68.1%).
n=414 cells.
40
Results
100
90
80
70
60
50
40
30
20
10
0
Figure 12. Occurrence of blebs in combination with a pseudovacuole. Left bar:
percentage of cells with a pseudovacuole near the bleb. Right bar: Percentage blebbing
cells lacking a pseudovacuole. n=150 cells. Error flag: mean ± standard deviation of 3
experiments.
41
Results
90
80
70
60
50
40
30
20
10
0
ctrl
5%
10%
Figure 13. Influence of hyperosmotic media on the occurrence of blebbing in Walker
carcinosarcoma cells. Error flag: mean ± standard deviation.
42
Results
Figure 14. Light scanning microscopic recording of an untreated, living Walker
carcinosarcoma cell. Figure 14A shows the cell in brightfield optics, with one or two
pseudovacuole(s) in longitudinal line with the cell. Figures 14B and 14C show
respectively the relative lack of nucleic acids (SYTO 17 stain) and hydrophobic content
(DiI stain) in the pseudovacuole, and the absence of a surrounding pseudovacuolar
membrane (inset). Figure 14D shows the fluorescent label pattern of CDFA SE of the
same cell. The lysine marker preferentially stains the pseudovacuole, confirming the
occurrence of the difficult to observe pseudovacuole in the brightfield picture.
P=pseudovacuole, N=nucleus, U=Uropod.
43
Results
Figure 15. Brightfield and CFDA SE labelled recordings of untreated, living Walker
carcinosarcoma cells at 37°C. Figures 15A shows a typical polarized cell with a clear colocalisation of the frontal pseudovacuole and the CFDA SE label. Figure 15B shows the
occurrence of a pseudovacuole in a spherical Walker carcinosarcoma cell and Figure 15C
shows a burst cell, with the pseudovacuole in the burst. B=burst, N=nucleus,
P=pseudovacuole, U=uropod.
44
Results
Figure 16. Transmission electron micrographs of chemical fixed and Epon embedded
untreated Walker carcinosarcoma cells. Figure 16A. Overview of a chemical fixed Walker
carcinoma cell. The cell appears as spherical, but this is depending on the sectioning
process. The tentative pseudovacuolar inclusions are visible as the highly electron
transparent features. Despite two inclusions are depicted, most probably they are two
regions of one continuous pseudovacuole, joined in another Z-axial plane. Figure 16B.
Enlargement of Figure 15A, showing the lack of a surrounding membrane as well as a
lacking content. N=Nucleus, Ne=Nuclear envelope, Nu=Nucleolus, P=Pseudovacuole.
45
Results
Figure 17. Transmission electron micrographs of high pressure frozen, freeze substituted
and Epon embedded Walker carcinosarcoma cells. Figure 17A. Overview of Walker
carcinoma cells at low magnification. Pseudovacuoles are observable as electron
transparent inclusions. Inset and figure 17B. Larger magnifications of a cell containing a
pseudovacuole. Membranes surrounding the pseudovacuoles are lacking (compare with
the membrane surrounding the vesicle). Despite the absence of membrane interfaces,
ribosomes and large protein complexes, visible as black globular entities, from the
cytoplasm are not present in the pseudovacuole.
46
Results
Figure 18. Co-localisation of the pseudovacuole as observed in light scanning microscopy
and the inclusions observed in transmission electron microscopy. The orientation of all
the figures is identical. The LSM recordings are taken 2-3 µm deeper along the z-axis.
LSM recordings show the distribution of CFDA SE label, a lysine marker. LM sections were
stained with toluidine blue. Figure 18A-C shows the same, nominated cell (arrowhead) in
(A) LSM, (B) LM and (C) TEM. 18A correlates the cell with 18B via the distance from the
edge of the sample, illustrated with the bars. 18C shows the overlap between 18B (LM)
and 18D, accentuate the exact mach of the cells. 18D TEM of high pressure frozen, FS
and resin embedded sample of Walker carcinosarcoma cells. The nominated cell (arrow)
has an inclusion along the right flank, which co-localizes with the CFDA SE stain
indicating a pseudovacuole of 18F (arrow). The presence of an uropod in 18F is due to
the 2-3 µm shift in the z-axis. 18E shows a larger magnification of the inclusion of 18D.
Note that the inclusion is not surrounded by a membrane. Bars: 20µm (A,B), 10µm (C),
5µm (D and F), 500nm (E).
47
Results
Figure 19. Transmission electron micrograph of a high pressure frozen, freeze substituted
and Epon embedded Walker carcinosarcoma cell. Onset of cortex detachment from the
cellular membrane is shown. C=cortex M=Mitochondria, N=Nucleus, V=vesicle.
48
Results
3.3. ADDITIONAL RESULTS
3.3.1 DEXTRAN TOXICITY
In order to find out if dextran was harmful for the cells before fixation, Walker
carcinosarcoma cells were grown in a medium containing different concentrations of
dextran (70 kda). 1 ml of a cell suspension with a density of 60.103 cells per ml (start
density of 60.102 cells/ml) was diluted in 9 ml RPMI1640 medium and the cells were
allowed to grow for 96 hours. The results are depicted in Figure 20. Up to a concentration
of 10 mg/ml dextran, growth was possible, and blebbing is not affected. Growing rates
and doubling times are summarized in Table 2. Although a lower growing rate and longer
doubling time are the result from the dextran in the medium, growth and cell division still
took place. Since the cells come only very shortly before fixation (< 1 minute) in contact
with dextran, I believe that dextran is not causing significant alterations.
Cell density (1000 of cells/ml)
250
cell count
blebbing
200
150
100
50
0
0
5
10
20
Dextran concentration (%)
Figure 20. Cell counts and blebbing cells after 96h at 37°C in RPMI1640 medium
containing different concentrations of dextran (70 kda).
Control
Growing rate
5.03 (±0.20)
Doubling time (h)
19
5 mg/ml
4.65 (±0.22)
21
10 mg/ml
1.95 (±0.54)
49
20 mg/ml
1.13 (±1.14)
85
Table 2. Growing rates and doubling time for cells grown in the presence of dextran (70
kda). The growing rate equals the number of times the density of the population doubled
(± standard deviation).
49
Results
3.3.2 THE MICROBIOPSY SYSTEM
Biopsy systems for sampling prior to high pressure freezing were developed (Hohenberg
et al., 1995; Shimoni and Müller, 1998) but they did not fully fulfil the needs as
described above. The system consists in three components: a biopsy gun, a biopsy
platelet and a transfer system. The system is described in detail in Vanhecke et al.
(2003) and Vanhecke et al. (2005).
The microbiopsy system was originally developed focussed on Mammalian tissue, but it
proved useful for fast sampling of cells in suspension too.
The biopsy gun
Three different shapes of the tip of the inner needle were produced in order to obtain the
highest efficiency in excision (Figure 21). The cavity was in all cases equal in size
(1.2mm x 0.6mm x 0.3mm). The size of the cavity was chosen small to avoid excessive
ice crystal formation during high pressure freezing, but large enough to obtain samples
without too much deformation due to excision.
0.5
1.2
0.6
0.3
1.2
1.5
0.6
0.3
1.3
2.1
1.2
0.6
0.3
Figure 21. shows illustrations (left) and schemes (right) of three different shape of the tip
of the inner needle of the microbiopsy gun. Top: blunt needle, middle: inverted needle,
bottom: straight needle. Measurements are in millimeter. The cavity has always a length
of 1.2 mm, a depth of 0.3 mm and a width of 0.6 mm.
Cow kidney was obtained from the local supermarket to test filling efficiency of the
different needle tips. In the case of the blunt needle 40% of the attempts resulted in a
filled needle cavity, while the inverted needle and the straight needle were filled in
respectively 85% and 100% (n=20) of the cases. The cavity of the blunt needle
contained large air bubbles in all cases. More than half of the cases showed gas bubbles
50
Results
when the inverted needle was used. Air bubbles did also occur in the case of the straight
needle (50% of the shots), but they were considerably smaller. All further experiments
were therefore carried out with the straight needle.
Sample holders and transfer system
A sample holder with a slot matching the cavity of the inner biopsy needle was developed
(Figure 22 left). The thickness of the sample (0.3mm) is to be considered rather thick to
obtain ice crystal free samples. However, excision of smaller samples resulted in the
disruption of the entire sample.
For fast sampling of cells in suspension, a copper ring was used with the microbiopsy
transfer system (Figure 22, right). Since the microbiopsy platelet has the same outer
dimensions as the copper ring, the latter one fits into the tools of the microbiopsy
transfer system. 5 µl5 of a 37°C warm cell pellet was pipetted in the central cavity of the
carrier and using the position tools of the microbiopsy transfer system placed and fixed
into a carrier for the high pressure freezer. The sampling protocol was finished within 20
seconds, and at every step, tools and sample were kept at 37°C.
3.0
3.0
1.2
0.3
0.3
1.2
0.2
0.6
Microbiopsy platelet
Copper ring
Figure 22. shows the microbiopsy platelet (left) with a cavity matching the cavity of the
inner needle of the microbiopsy gun and freeze fracture carriers (right) with the similar
diameter but a reduced platelet thickness (0.6mm vs 0.2mm). Values are in millimetre.
3.3.3 ALTERNATIVE SAMPLING OF CELL SUSPENSIONS
The copper tube system for the Leica EMPACT is a suitable system for high pressure
freezing of cell suspensions and yields in general excellent results. The copper tubes are
600 µm thick, with a wall thickness of 140 µm. A drop of five µl of cell pellet is pipetted
onto a clean formvar. The copper tube, hold in a therefore designed carrier, is filled using
a wire of Ø 0.25mm as a piston (Figure 23). Sampling using this method took
approximately 20 seconds.
5
The copper ring cavity has a volume of about 0.22 µm3. Surface tension phenomena make it impossible to fill
the ring cavity and excess pellet is used.
51
Results
Figure 23. Left: Filling a copper tube with a cell suspension using a wire as piston. Right:
SEM picture of a sliced open copper tube (bar=100µm)
52
References
4. References
1.
Abercrombie, M., Heaysman, J.E.M, Pegrum, S.M. (1970a) The locomotion of fibroblasts in culture. I.
Movements at the leading edge. Experimental cell research 59: 393-398.
2.
Abercrombie, M., Heaysman, J.E.M, Pegrum, S.M. (1970b) The locomotion of fibroblasts in culture. II.
Ruffling. Experimental cell research 60: 437-444.
3.
Abercrombie, M., Heaysman, J.E.M, Pegrum, S.M. (1970c) The locomotion of fibroblasts in culture. III.
Movements of particles on the dorsal surface of the leading lamella. Experimental cell research 62: 389398.
4.
Abercrombie, M., Heaysman, J.E.M, Pegrum, S.M. (1971) The locomotion of fibroblasts in culture. IV.
Electron microscopy of the leading edge. Experimental cell research 67: 359-367.
5.
Abercrombie, M., Heaysman, J.E.M, Pegrum, S.M. (1972) The locomotion of fibroblasts in culture. V
Surface marking with concanavalin A. Experimental cell research 73: 536-539.
6.
Adrian, M., Dubochet, J., Lepault, J. & McDowall, A. W. (1984) Cryo-electron microscopy of viruses.
Nature 308: 32-36.
7.
Al-Amoudi, A., Chang, J.J., Leforestier, A., McDowall, A., Salamin, L.M., Norlen, L.P.O., Richter, K.,
Blanc, N.S., Studer, D., Dubochet, J. (2004a) Cryo-electron microscopy of vitreous sections. EMBO
journal 23 (18): 3583-3588.
8.
Al-Amoudi, A., Norlen, L.P.O., Dubochet, J. (2004b) Cryo-electron microscopy of vitreous sections of
native biological cells and tissues. Journal of structural biology 148(1): 131-135.
9.
Albrecht-Buehler, G. (1982) Does blebbing reveal the convulsive flow of liquid and solutes through the
cytoplasmic meshwork? Cold spring harbor symposia on quantitive biology XLVI: 45-49.
10.
Anderson, K.I., Wang, Y-L., small, J. (1996) Coordination of protrusion and translocation of the
11.
Anderson K.I. and Cross, R. (2000) Contact dynamics during keratocyte motility. Current biology 10:
keratocyte involves rolling of the cell body. Journal of cell biology 134(5): 1209-1218.
253-260.
12.
Angell, C.A. and Choi, Y. (1986) Crystallization and vitrification in aqueous systems. Journal of
microscopy 141(3): 251-261.
13.
Bachmann, L. & Mayer, E. (1987) Physics of water and ice: implications for cryofixation. Cryotechniques
in Biological Electron Microscopy (ed. By R.A. Steinbrecht and K. Zierold), pp. 3-37. Springer-Verlag,
Berlin.
14.
Bereiter-Hahn, J., Strohmeier, R., Knuzenbacher, I., Beck, K., Voth, M. (1981) Locomotion of Xenopus
epidermis cells in primary culture. Journal of cell science 52: 289-311.
15.
Bereiter-Hahn, J. (1985) Architecture of tissue cells: the structural basis which determines shape and
locomotion of cells. Acta Biotheoretica 34: 139-148.
16.
Biel, S.S., Kawaschinski, K., Wittern, K.P., Hinze, U., Wepf, R. (2003) From tissue to cellular
ultrastructure: closing the gap between micro- and macrostructural imaging. Journal of microscopy
212(1): 91-99.
17.
Bohrmann, B. and Kellenberger, E. (2001) Cryosubstitution of frozen biological specimens in electron
microscopy: use and application as an alternative to chemical fixation. Micron 32:11-19.
18.
Boulbitch, A., Simson, R., Simson, D.A., Merkel, R., Häckl, W., Bärmann, M., Sackmann, E. (2000)
Shape instability of a biomembrane driven by a local softening of the underlying actin cortex. Physical
review E 62(3): 3974-3985.
53
References
19.
Bowers, B. and Maser, M. (1988) Artifacts in fixation for transmission electron microscopy. In: Artifacts
in biological electron microscopy (Crang, R.F.E. and Klomparens, K.L., eds), Plenum press, NY and
London.
20.
Boyde, A. and Maconnachie, E. (1979) Volume changes during preparation of mouse embryonic tissue
21.
Bretscher, M.S. (1996) Getting membrane flow and cytoskeleton to cooperate in moving cells. Cell 87:
for scanning electron microscopy. Scanning 2: 149-163.
601-606.
22.
Burton, K., Park, J.H., Taylor, D.L. (1999) Keratocytes generate traction forces in two phases. Molecular
biology of the cell 10: 3745-3769.
23.
Butz, P., Ries, J., Traugott, U., Weber, H., Ludwig, H. (1990) Hochdruckinactivierung von Bacterien und
24.
Butz, P. and Ludwig, H. (1991) Hochdruckinaktivierung von Hefen und Schimmelpilzen. Pharmazeutische
Bakteriensporen. Pharmazeutische Industrie 52(4): 487-491.
Industrie 53(6): 584-586
25.
Cameron, I.L. (1988) Ultrastructural observations of the transectioned end of frog skeletal muscle cells.
Physiological chemistry and physics and medical NMR 20: 221-225.
26.
27.
Carlslaw H.S. and jaeger, J.C. (1959) conduction of heat in solids. 2nd ed. Clarendon Press. Oxford.
Carr, D.W., Stofko-Hahn, R.E., Fraser, I.D., Bishop, S.M., Acott, T.S., Brennan, R.G., Scott, J.D. (1991)
Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins
occurs through an amphipathic helix binding motif. Journal of biological chemistry 266(22): 1418814192.
28.
Chang, D.C. and Reese, T.S. (1990) Changes in membrane structure induced by electroporation as
29.
Chaplin, M. (2005) URL: http://www.lsbu.ac.uk/water/
30.
Claeys M, Vanhecke D, Couvreur M, Tytgat T, Coomans A, Borgonie G (2004) High pressure freezing and
revealed by rapid-freezing electron microscopy. Biophysical journal 58: 1-12.
freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron
microscopy. Nematology 6(3):319-327.
31.
Clegg, J.S. (1988) Contrasting views on the organization of cytoplasm. Scanning microscopy 2(2): 105-
32.
Condeelis, J. (1993) Life at the leading edge: the formation of cell protrusions. Annual reviews in cell
114.
biology 9: 411-444.
33.
Cox, D., Ridsdale, J.A., Condeelis, J., Hartwig, J. (1995) Genetic deletion of ABP-120 alters the 3dimensional organization of actin-filaments in Dictyostelium pseudopods. Journal of cell biology, 128(5):
819-835.
34.
Cramer, L.P. (1999) Role of actin-filament disassembly in lamellipodium protrusion in motile cells
revealed using the drug jasplakinolide. Current biology 9: 1095-1105.
35.
Crews, P., Manes, L.V., Boehler, M. (1986). Jasplakinolide, a cyclodepsipeptide from the marine sponge,
jaspis sp. Tetrahedron letters 27(25): 2797-2800.
36.
Cunningham, C.C., Gorlin, J.B., Kwiatkowski, D.J., Hartwig, J.H., Janmey, P.A., Byers, H.R., Stossel, T.P.
(1992) Actin-binding protein requirement for cortical stability and efficient locomotion. Science
255(5042): 325-327.
37.
Cunningham, C.C. (1995) Actin polymerization and intracellular solvent flow in cell surface blebbing.
38.
Dahl, R. and Staehelin, L.A. (1989) High-pressure freezing for the preservation of biological structure:
Journal of cell biology 129(6): 1589-1599.
theory and practice. Journal of electron microscopy technique 13: 165-174.
39.
De Broglie, M.L. (1923) Radiations – Ondes et quanta. Comptes rendus de l'académie des sciences 177:
507-510.
40.
Dembo, M. (1989) Mechanics and control of the cytoskeleton in Amoeba proteus. Biophysical journal 55:
1053-1080.
54
References
41.
Ding, B., Turgeon, R., Parthasarathy, M.V. (1991) Effect of high pressure freezing on plant filament
bundles. Journal of microscopy 165(3): 367-376.
42.
Ding, B., Turgeon, R., Parthasarathy, M.V. (1992) Structure of freeze-substituted plasmodesmata.
Protoplasma 169: 28-41.
43.
Dipasquale, A. (1975) Locomotion of epithelial cells: factors involved in the extension of the leading
edge. Experimental cell research 95: 425-439.
44.
Diviani, D., Soderling, J., Scott, J.D. (2001) AKAP-Lbc anchors protein kinase A and nucleates Galpha 12selective Rho-mediated stress fiber formation. Journal of biological chemistry 276(47): 44247-44257.
45.
Dubochet, J., Adrian, M., Chang, J.-J., Homo, J.-C., Lepault, J., McDowall, A. W. & Schultz, P. (1988),
Cryo-electron microscopy of vitrified specimens. Quaterly review of biophysics 21: 129-228.
46.
Echlin, P. (1992) Low-temperature microscopy and analysis. Plenum Press, NY and London. 539p.
47.
Edelmann, L. (1986) Two opposing theories of the cell: experimental testing by cryomethods and
electron microscopy. In Science of biological specimen preparation (Müller, M., Becker, R.P., Boyde, A.,
Wolosewick, J.J., eds.) SEM inc. AMF O’Hare, Chicago, IL 33-43.
48.
Edelmann, L., (1988) The cell water problem posed by electron microscopic studies of ion binding in
muscle. Scanning microscopy 2(2): 851-865.
49.
Edelmann, L. (1978) A simple freeze-drying technique for preparing biological tissue without chemical
fixation for electron microscopy. Journal of microscopy 112(2): 243-248.
50.
Eggli, P.S., Graber, W. (1994) Improved ultrastructural preservation of rat ciliary body after high
pressure freezing and freeze substitution: a perspective view based upon comparison with tissue
processed according to conventional protocol or by osmium tetroxide/microwave fixation. Microscopy
research and technique 29: 11-22.
51.
Escaig, J. (1982) New instruments which facilitate rapid freezing at 83K and 6K. Journal of microscopy
126(3): 221-229.
52.
Feder, N. and Sidman, R.L. (1958) Methods and principles of fixation by freeze-substitution. Journal of
biophysical and biochemical cytology 4(5): 593-600.
53.
Fedier, A. and Keller, H.U. (1997) Suppression of bleb formation, locomotion and polarity of Walker
carcinosarcoma cells by hypertonic media correlates with cell volume reduction but not with changes in
the F-actin content. Cell motility and the cytoskeleton 37: 326-337.
54.
Fedier, A., Eggli, P., Keller, H.U. (1999) Redistribution of surface-bound Con A is quantitatively related to
55.
Fernández-Morán, H. and Dahl, A.O. (1952) Electron microscopy of ultrathin frozen sections of pollen
the movement of cells developing polarity. Cell motility and cytoskeleton 44: 44-57.
grains. Science 116(3018): 465-467.
56.
Friedl, P. and Bröcker, E.B. (2000) The biology of cell locomotion within three-dimensional extracellular
matrix. Cellular and molecular life sciences 57: 41-64.
57.
Gabriel, D., Hacker, U., Köhler, J., Müller-Taubenberger, A., Schwartz, J.M., Westphal, M., Gerisch, G.
(1999) The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized
by a GFP marker protein. Journal of cell science 112(22): 3995-4005.
58.
Gilkey, J.C., and Staehelin, L.A. (1986) Advances in ultrarapid freezing for the preservation of cellular
structure. Journal of electron microscopy technique 3: 177-210.
59.
Grebecka, L., Grebecki, A. (1981) Testing motor functions of the frontal zone in the locomotion of
60.
Grebecki, A. (1994) Membrane and cytoskeleton flow in motile cells with emphasis on the contribution of
Amoeba proteus. Cell biology international reports 5(6): 587-594.
free-living amoebae. International review of cytology 148: 37-80.
61.
Gross, H. (1987) High resolution metal replication of freeze dried specimens. In: Cryotechniques in
biological electron microscopy, Steinbrecht, R.A. and Zierold, K. (eds), Springer-Verlag, Berlin.
62.
Gu, L., Zhang, H., Chen, Q., Chen, J. (2003) Calyculin A-induced actin phosphorylation and
depolymerization in renal epithelian cells. Cell motility and cytoskeleton 54: 386-295.
55
References
63.
Hagmann, J., Burger, M.M., Dagan, D. (1999) Regulation of plasma membrane blebbing by the
cytoskeleton. Journal of cellular biochemistry 79: 488-499.
64.
Hayat, M.A. (1986) Basic techniques for transmission electron microscopy. Academic press. 411p.
65.
Hayat, M.A. (2000) Principles and techniques of electron microscopy, biological applications (4th edition).
Cambridge university press. 543p.
66.
Hazlewood, C.F. and Kellermayer, M. (1988) Ion and water retention by permeabilized cells. Scanning
microscopy 2(2): 17-22.
67.
Heidemann, S.R. and Bixbaum, R., E. (1998) Cell crawling: first the motor, now the transmission.
Journal of cell biology 141(1): 1-4.
68.
Henderson (1995) Henderson’s dictionary of biological terms (11th edition). Lawrence, E., ed. Longman
group limited. 693p.
69.
Hernandez-Verdun, D., Quitana, C., Masson, C., Gautier, T., Arnoult, J. (1991) Cryofixation,
cryosubstitution, cryo-embedding for visualizing of nuclear ultrastructure and for immunodetection in
HeLa cells. Biology of the Cell 72: 121-132.
70.
Hunziker, E.B., Michel, M., Studer, D. (1997) Ultrastructure of adult human articular cartilage matrix
after cryotechnical processing. Microscopy research and technique 37: 271-284.
71.
Jakstys, B.P. (1988) Artifacts in sampling specimens for biological electron microscopy. In: Artifacts in
biological electron microscopy (Crang, R.F.E. and Klomparens, K.L., eds), Plenum press, NY and London.
72.
Janson. L.W. and Taylor, D.L. (1993) In vitro models of tail contraction and cytoplasmic streaming in
amoeboid cells. Journal of cell biology 123(2): 345-356.
73.
Jimenez, C., Portela, R.A., Mellado, M., Rodriguez-Frade, J.M., Collard, J., Serrano, A., Martinez, A.C.,
Avila, J., Carrera, A.C. (2000) Role of the PI3K regulatory subunit in the control of actin organization and
cell migration. Journal of cell biology 151(2): 249-62.
74.
Jones, G.E. (2000) Cellular signaling in macrophage migration and chemotaxis. Journal of leukocyte
75.
Jung, H.I., Shin, I., Park, Y.M., Kang, K.W., Ha, K.S. (1997) Colchicine activates actin polymerization by
Biology 68(5): 593-602.
microtubule depolymerisation. Molecular cells 7(3): 431-437.
76.
Kaibuchi, K., Kuroda, S., Amano, M. (1999) Regulation of the cytoskeleton and cell adhesion by the Rho
family GTPases in Mammalian cells. Annual reviews in biochemistry 68: 459-486.
77.
Kanno, H., Speedy, R.J., Angell, C.A. (1975) Supercooling of water to –92°C under pressure. Science
189: 880-881.
78.
Kellenberger, E. (1987) The response of biological macromolecules and supramolecular structures to the
physics of specimen cryopreparation. In: Cryotechniques in biological electron microscopy, Steinbrecht,
R.A. and Zierold, K. (eds), Springer-Verlag, Berlin.
79.
Keller, H.U. and Bebie, H. (1996) Protrusive activity quantitatively determines the rate and direction of
80.
Keller, H.U. and Eggli, P. (1998a) Protrusive activity, cytoplasmic compartmentalization, and restriction
locomotion. Cell motility and cytoskeleton 33: 241-251.
rings in locomoting blebbing Walker carcinosarcoma cells are related to detachment of cortical actin from
the plasma membrane. Cell motility and the cytoskeleton 41: 181-193.
81.
Keller, H.U. and Eggli, P. (1998b) Actin accumulation in pseudopods or in the tail of polarized Walker
carcinosarcoma cells quantitatively correlates with local folding of the cell surface membrane. Cell
motility and the cytoskeleton, 40: 342-353.
82.
Keller, H.U. (2000) Redundancy of lamellipodia in locomoting Walker carcinosarcoma cells. Cell motility
and the cytoskeleton 46: 247-256.
83.
Keller, H.U., Dehghani Zadeh, A., Eggli, P. (2002a) Localized depletion of polarized actin at the front of
Walker carcinosarcoma cells increases the speed of locomotion. Cell motility and the cytoskeleton 53:
189-202.
56
References
84.
Keller, H.U., Rentsch, P., Hagmann, J. (2002b) Differences in cortical actin structure and dynamics
document that different types of blebs are formed by distinct mechanisms. Experimental cell research
277: 161-172.
85.
Kern, W. (1948) Die Activität der Natriumionen in wäβrigen Lösungen der Salze mit polyvalenten Säuren.
86.
Kiss, J.Z., Giddings Jr., T.H., Staehelin, L.A., Sack, F.D. (1990) Comparison of the ultrastructure of
Makromolekulare chemie 2:279-288.
conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis.
Protoplasma 157: 64-74.
87.
Klemke, R.L., Cai, S., Giannini, A.L., Gallagher, P.J., deLanerolle, P., Cheresh, D.A. (1997) Regulation of
cell motility by mitogen-activated protein kinase. Journal of cell biology 137(2): 481-492.
88.
Klenchin, V.A., Sukharev, S.L., Serov, S.M., Chernomordik, L.V., Chizmadzhev, Y.A. (1991) Electrically
induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophysical journal 60: 804811.
89.
Knoll, M. and Ruska, E. (1932) Das Elektronenmikroscop. Zeitschrift für physik 78: 318-339.
90.
Kole, T.P., Tseng, Y., Jiang, I., Katz, J.L., Wirtz, D. (2005) Intracellular mechanics of migrationg
fibroblasts. Molecular biology of the cell 16: 328-338.
91.
Korohoda, W., Drukala, J., Sroka, J., Madeja, Z. (1997) Isolation, spreading, locomotion on various
substrata, and the effect of hypotonicity on locomotion of fish keratinocytes. Biochem. Cell biology 75:
277-286.
92.
Krause, T.L., Fishman, H.M., Ballinger, M.L., Bittner, G.D. (1994) Extent and mechanism of sealing in
transected giant axons of squid and earthworms. Journal of neuroscience 14: 6638-6651.
93.
Lauffenberger, D.A. and Horwitz, A.F. (1996) Cell migration: a physically integrated molecular process.
Cell 84: 359-369.
94.
Lee, R.M.K.W., McKenzie, R., Kobayashi, K., Garfield, R.E., Forrest, J.B., Daniel, E.E. (1982) Effects of
glutaraldehyde fixative osmolarities on smooth muscle cell volume, and osmotic reactivity of the cells
after fixation. Journal of microscopy 125(1): 77-88.
95.
Lee, J., Ishihara, A., Theriot, J.A., Jacobsen, K. (1993) Principles of locomotion for simple-shaped cells.
96.
Leforestier, A., Richter, K., Livolant, F., Dubochet, J. (1996) Comparison of slam-freezing and High-
Nature, 362: 167-171.
ressure freezing effects on the DNA cholesteric liquid crystalline structure. Journal of microscopy 184(1):
4-13.
97.
Ling, G.N. (1952) The role of phosphate in the mainenance of the resting potential and selective ionic
accumulation in frog muscle cells. in: Phosphorous metalbolism Vol. 2 (eds: W.D. McElroy and B. Glass).
The John Hopkins University Press, Baltimore, US. 748-797.
98.
Ling, G.N. (1960) The Interpretation of Selective Ionic Permeability and Cellular Potentials in Terms of
the Fixed Charge-Induction Hypothesis. Journal of general physiology 43 (5): 149-174.
99.
Ling, G.N. (1962) A physical theory of the living state: the association-induction hypothesis. Blaisdell
publishing, NY and London. 680p.
100.
Ling, G.N. and Ochsenfeld, M.M. (1966) Studies on ion accumulation in muscle cells. Journal of general
physiology 49: 819-843.
101.
Ling, G.N. and Zhang, Z.L. (1983a) Evidence that Na+ in a sulfonate ion exchange resin exists in an
adsorbed state. Its significance for the interpretation of NMR data in resins and cells. Physiological
chemistry and physics and medical NMR 15: 251-258.
102.
Ling, G.N. and Zhang, Z.L. (1983b) Studies on the physical state of water in living cells and model
systems IV. Freezing and thawing point depression of water by gelatin, oxygen-containing polymers and
urea-denaturated proteins. Physiological chemistry and physics and medical NMR 15: 391- 406.
103.
Ling, G.N. and Zhang, Z.L. (1984) A study of selective adsorption on Na+ and other alkali-metal ions on
isolated proteins: a test of the salt-linkage hypothesis. Physiological chemistry and physics and medical
NMR 16: 221-235
57
References
104.
Ling, G.N. and Ochsenfeld, M.M. (1991) The majority of potassium ions in muscle cells is adsorbed on βand γ-carboxyl groups of myosin: potassium-ion adsorbing carboxyl groups on myosin heads engage in
cross-bridge formation during contraction. Physiological chemistry and physics and medical NMR 23:
133-160.
105.
Ling, G.N. (1992) A revolution in the physiology of the living cell. Krieger publishing company, Malabar,
106.
Ling G.N. (2001) Life at the cell and below cell level: the hidden history of a fundamental revolution in
FL, USA. 378p
biology. Pacific press, New York, US. 373p.
107.
Mast. S.O. (1926) Structure, movement, locomotion, and stimulation in Amoeba. Journal of morphology
and physiology 41(2): 347-424.
108.
Manning, G.S. (1978) The molecular theory of polyelectrolyte solutions with applications to the
electrostatic properties of polynucleotides. Quarterly reviews of biophysics 2: 179-246.
109.
Macdonald, A.G. (1984) The effects of pressure on the molecular structure and physiological functions of
cell membranes. Phil. Trans. R. Soc. Lond. B 304: 47-68.
110.
McDonald, K., Morphew, M.K. (1993) Improved preservation of ultrastructure in difficult-to-fix organisms
by high pressure freezing and freeze substitution: I. Drosophila melanogaster and strongylocentrotus
purpuratus embryos. Microscopy research and technique 24: 465-473
111.
Merkel, R., Simson, R., Simson, D.A., Hohenadl, M., Boulbitch, A., Walraff, E., Sackmann, E. (2000) A
micromechanic study of cell polarity and plasma membrane cell body coupling in Dictyostelium.
Biophysical journal 79: 707-719.
112.
Mitchison, T.J. and Cramer, L.P. (1996) Actin-based cell motility and cell locomotion. Cell 84: 371-379
113.
Monaghan, P., Perusinghe, N. & Müller, M. (1998) High pressure freezing for immunocytochemistry.
Journal of microscopy 192(3): 248-258.
114.
Moor, H. (1987) Theory and practice of high pressure freezing. Cryotechniques in Biological Electron
115.
Moor, H. and Hoechli, M. (1970) The influence of high pressure freezing on living cells. In: Microscopy
Microscopy (ed. By R.A. Steinbrecht and K. Zierold), pp. 175-191. Springer-Verlag, Berlin.
electronique, Favard, P. (ed), Proceedings of the 7th international congress on electron microscopy. 445446.
116.
Müller, M. (1988) Cryopreparation of microorganisms for electron microscopy. Methods in microbiology
20: 1-27.
117.
Nakanishi, S., Yamada, K., Iwahashi, K., Kuroda, K., Kase, H. (1990) KT5926, a potent and selective
inhibitor of myosin light chain kinase. Molecular pharmacology 37(4): 482-488.
118.
119.
Negendank, W. (1988) The state of water in the cell. Scanning microscopy 2(2): 41-52.
Nickels, J. (1970) Localization of a microelectrode tip in muscle cell: a light and electron microscopic
study. Acta physiologica scandinavica 80: 360-369.
120.
Niggli, V. (2003) Signaling to migration in neutrophils: importance of localized pathways. International
121.
Oliver, T., Dembo, M., Jacobson, K. (1995) Traction forces in locomoting cells. Cell motility and the
122.
Oliver, T., Dembo, M., Jacobson, K. (1999) Separation of propulsive and adhesive traction stresses in
journal of biochemistry and cell biology 35: 1619-1638.
cytoskeleton 31: 225-240.
locomoting keratocytes. Journal of cell biology 145(3): 589-604.
123.
Ostap, E.M. (2002) 2,3-Butanedione monoxime (BDM) as a myosin inhibitor. Journal of muscle research
and cell motility 23: 305-308.
124.
Oster, G.F. (1984) On the crawling of cells. Journal of embryology and experimental morphology 83
supplement: 329-364.
125.
Oster, G.F. and Perelson, A.S. (1987) The physics of cell motility. Journal of cell science supplement 8:
35-54.
58
References
126.
Paku, S., Tóvári, J., Lötincz, Z., Timár, F., Döme, B., Kopper, L., Raz, A., Tímár, J. (2003) Adhesion
dynamics and cytoskeletal structure of gliding human fibrosarcoma cells: a hypothetical model of cell
migration. Experimental cell research 290: 246-253.
127.
Palecek, S.P., Schmidt, C.E., Lauffenburger, D.A., Horwitz, A.F. (1996) Integrin dynamics on the tail
region of migrating fibroblasts. Journal of cell science 109(5): 941-952.
128.
Peckham, M., Miller, G., Wells, C., Zicha, D., Dunn, G. (2001) Specific changes to the mechanism of cell
locomotion induced by overexpression of β-actin. Journal of cell science, 114: 1367-1377.
129.
Plattner, H., Bachmann, L. (1982) Cryofixation: a tool in biological ultrastructural research. International
review of cytology 79: 237-305.
130.
Pollack, G.H. (2001) Cells, gels and the engines of life: a new unifying approach to cell function. Ebner
131.
Rafelski, S.M. and Theriot, J.A. (2004) Crawling toward a unified model of cell motitliy: spatial and
Publishers, Seattle, WA, USA.
temporal regulation of actin dynamics. Annual reviews in biochemistry. 73: 209-239.
132.
Rentsch, P.S., Keller, H.U. (2000) Suction pressure can induce uncoupling of the plasma membrane from
cortical actin. European journal of cell biology 79: 975-981.
133.
Ridley, A..J. (2001) Rho GTPases and cell migration. Journal of Cell Science. Vol 114(Pt 15):2713-2722
134.
Riehle, U. and Hoechli, M. (1973 ) The theory and technique of high pressure freezing. In: freeze-etching
technique and applications (Benedetti, E.C. and Favard, P., eds) p31-61.
135.
Riento, K. and Ridley, A.J. (2003) ROCKS: multifunctional kinases in cell behaviour. Nature reviews in
136.
Robards, A.W. and Sleytr, U.B. (1985) Low temperature methods in biological electron microscopy.
137.
Royer, S.M., Kinnamon, J.C. (1996) Comparison of high-pressure freezing/freeze substitution and
molecular cell biology 4: 446-456.
Elsevier publishing, Amsterdam.
chemical fixation of catfish barbell taste buds. Microscopy research and technique 35: 385-412.
138.
Rostaing, P., Weimer, R.M., Jorgensen, E.M., Triller, A., Bessereau, J.L. (2004) Preservation of
immunoreactivity and fine structure of adult C. elegans tissue using high-pressure freezing. Journal of
histochemistry and cytochemistry 52(1): 1-12.
139.
Ruska, E. and Knoll, M. (1931) Die magnetische Sammelspule für schnelle Elektronenstrahlen. Zeitschrift
für technische physik 12: 389-400.
140.
Sartori, N., Richter, K. & Dubochet, J. (1993a) Vitrification depth can be increased more than 10-fold by
141.
Sartori, N., Richter, K. & Dubochet, J. (1993b) High pressure freezing increases the vitrification depth.
high pressure freezing. Journal of microscopy 172(1): 55-61.
Experimentia 49: A41
142.
Schmidt, A. and Hall, M.N. (1998) Signaling to the actin cytoskeleton. Annual reviews in cellular
developmental biology 14: 305-338.
143.
Shousboe, P. (2003) Sucrose inhibits vascular smooth muscle cell proliferation/migration – protein
synthesis maintained. Cell proliferation 36: 55-61.
144.
Schütz, K., Keller, H.U. (1998) Protrusion, contraction and segregation of membrane components
associated with passive deformation and shape recovery of Walker carcinosarcoma cells. European
journal of cell biology 77: 100-110.
145.
Sells, M.A., Knaus, U.G., Bagrodia, S., Ambrose, D.M., Bokoch, G.M., Chernoff, J. (1997) Human p21-
146.
Semmler, K., Wunderlich, J., Richter, W., Meyer, H.W. (1998) High-pressure freezing causes structural
activated kinase (Pak1) regulates actin organization in mammalian cells. Current Biology 7(3): 202-210
alterations in phospholipid model membranes. Journal of microscopy 190(3): 317-327.
147.
Sheetz, M.P., Wayne, D.B., Pearlman, A.L. (1992) Extension of filopodia by motor-dependent actin
assembly. Cell motility and cytoskeleton 22: 160-169.
148.
Sheetz, M.P., Felsenfeld, D., Galbraith, C.G., Choquet, D. (1999) Cell-migration as a five-step cycle. In:
Cell behaviour: control and mechanism of motility (Lackie, J.M., Dunn, G.A., Jones, G.E., eds), Portland
press.
59
References
149.
Shimoni, E. & Müller, M. (1998) On optimising high pressure freezing: from heat transfer theory to a new
microbiopsy device. Journal of microscopy 192(3): 236-247.
150.
Sitte, H., Edelmann, L., Neumann, K. (1987) Cryofixation without pretreatment at ambient pressure. In:
Cryotechniques in biological electron microscopy, Steinbrecht, R.A. and Zierold, K. (eds), SpringerVerlag, Berlin.
151.
Skepper, J.N. (2000) Immunocytochemical strategies for electron microscopy: choice or compromise.
Journal of microscopy 199(1): 1-36.
152.
Small, V., Rottner, K., Kaverina, I. (1999) Functional design in the actin cytoskeleton. Current opinion in
cell biology 11: 54-60.
153.
Sroka, J., Von Gunten, M., Dunn, G.A., Keller, H.U. (2002) Phenotype modulation in non-adherent and
adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules
in controlling cell shape, locomotion and cytoskeletal structure. International journal of biochemistry and
cell biology 34: 882-899.
154.
Stedman (2000) Stedman’s medical dictionary (27th edition). Lippincott Williams & Wilkins.
155.
Stossel, T.P., Hartwig, J.H., Janmey, P.A., Kwiatkowski, D.J. (1999) Cells crawling two decades after
Abercrombie. In: Cell behaviour: control and mechanism of motility (Lackie, J.M., Dunn, G.A., Jones,
G.E., eds), Portland press.
156.
Strohmeier, R. and Bereiter-Hahn, J. (1987) Hydrostatic pressure in epidermal cells is dependent on Ca-
157.
Studer, D., Michel, M., Müller, M. (1989) High pressure freezing comes of age. Scanning microscopy
mediated contractions. Journal of cell science, 88(5): 631-640.
supplement 3: 253-269.
158.
Studer, D., Hennecke, H., Müller, M. (1992) High-pressure freezing of soybean nodules leads to an
improved preservation of ultrastructure. Planta 188: 155-163.
159.
Studer, D., Michel, M., Wohlwend, M., Hunziker, E.B. & Buschmann, M. (1995) Vitrification of articular
160.
Studer, D., Graber, W., Al-Amoudi, A. & Eggli, P. (2001) A new approach for cryofixation by high
cartilage by high-pressure feezing. Journal of microscopy 179(3): 321-332.
pressure freezing. Journal of microscopy 203(3): 285-294.
161.
Sun, Q., Zheng, H., Xu, J., Hines, E. (2003) Raman spectroscopic studies of the stretching band from
water up to 6 kbar at 290 K. Chemical physics letters 379: 427-431.
162.
Svitkina, T.M., Verkhovsky, A.B., McQuide, K.M., Borisy, G.G (1997) Analysis of actin-myosin II system
in fish epidermal keratocytes: mechanism of cell body translocation. Journal of cell biology 139(2): 397415.
163.
Taylor, L.D., Wang, Y-L., Heipe, J. (1980) Contractile basis of amoeboid movement VII. The distribution
164.
Thoumine, O. and Cardoso, O. (1999) Changes in the mechanical properties of fibroblasts during
165.
Thuret, G., Chiquet, C., Herrag, S., Dumollard, J.M., Boudard, D., Bednarz, J., Campos, L., Gain, P.
of fluorescently labeled actin in amoebas. Journal of cell biology 86: 590-598.
spreading: a micromanipulation study. European biophysical journal 28: 222-234.
(2005) Mechanisms of stauroporine induced apoptosis in a human corneal endothelian cell line. British
journal for ophthalmology 87: 346-352.
166.
Tokuyasu, K.T. (1973) A technique for ultracryotomy of cell suspensions and tissues. Journal of cell
biology 57: 551-565.
167.
Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa, M., Narumiya, S. (2000) Pharmacological
properties of Y-27632, a specific inhibitor of Rho-associated kinases. Molecular pharmacology 57(5):
976-983.
168.
Urwyler, N., Eggli, P., Keller, H.U. (2000) Effects of the myosin inhibitor 2,3-butanedione monoxime
(BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes. Cell
biology international 24(12): 863-870.
169.
Van Harreveld, A. & Crowell, J. (1964) Electron microscopy after rapid freezing on a metal surface and
substitution fixation. Anatomical record 149: 381-386.
60
References
170.
Van Harreveld, A., Crowell, J. & Malhotra, S.K. (1965) A study of extracellular space in central nervous
tissue by freeze-substitution. Journal of cell biology 25: 117-137.
171.
Vanhecke D, Graber W, Herrmann G, Al-Amoudi A, Eggli P, Studer D. (2003) A rapid microbiopsy system
to improve the preservation of biological samples prior to high-pressure freezing. Journal of microscopy
212(1): 3-12.
172.
Vanhecke, D., Eggli, P., Graber, W., Studer, D. (2005) A new microbiopsy system enables rapid
preparation of tissue for high pressure freezing. In Methods in molecular biology (Taatjes DJ, Ed.).
Humana press NY, USA. In press
173.
Von Mentrup, E., Butz, P., Stricker, H., Ludwig, H. (1988) Hochdrucksterilisation von Liposomen
Pharmazeutische Industrie 50(3): 363-366.
174.
Von Schack, M.L., Fakan, S., Villiger, W., Müller, M. (1993) Cryofixation and cryosubstitution: a useful
alternative in the analysis of cellular fine structure. European journal of histochemistry 37: 5-18.
175.
Von Tscharner Biino, N., Portzig, H., Keller, H.U. (1997) Suppression of polarity, locomotion and F-actin
levels of Walker carcinosarcoma cells by the inhibitor CI-959. Life sciences 61(2): 137-154.
176.
Wang, Y-L. (1985) Exchange of actin subunits at the leading edge of living fibroblasts: possible role of
treadmilling. Journal of cell biology 101: 597-602.
177.
Wantanabe, Y., Ito, M., Katoaka, Y., Wada, H., Koyama, M., Feng, J. (2001) Protein kinase C-catalysed
phosphorylation of an inhibitory phosphoprotein of myosin phosphatase is involved in human platelet
secretion. Blood 87(12): 3798-3805.
178.
Waterman-Storer, C.M. and Salmon, E.D. (1999) Positive feedback interactions between microtubule and
actin dynamics during cell motility. Current opinion in cell biology 11: 61-67.
179.
Watt, I.M. (1997) The principles and practice of electron microscopy (2nd edition). Cambridge academic
press. 484p.
180.
Wehrle-Haller, B., Imhof, B.A. (2002) The inner lives of focal adhesions. Trends in cell biology 12(8):
181.
Wicki, A. and Niggli, V. (1999) Role of protein kinase C isoforms in locomotion of Walker 256
382-389.
carcinosarcoma cells. International journal of cancer 81: 255-261.
182.
Wicki, A. and Niggli, V. (2001) The rho/rho-kinase and the phosphatidylinositol 3-kinase pathways are
essential for spontaneous locomotion of Walker carcinosarcoma 256 cells. International journal of cancer
91: 763-771.
183.
Wischnitzer, S. (1981) Introduction to electron microscopy, 3th edition. Pergamon Press. 405p
184.
Xie, T.D., Sun, L., Tsong, T.Y. (1990) Studies of mechanisms of electric field-induced DNA transfection.
Biophysical journal 58: 13-19.
185.
Yanai, M., Kenyon, C.M., Butler, J.P., Macklem, P.T., Kelly, S.M. (1996) Intracellular pressure is a motive
force for cell motion in Amoeba proteus. Cell motility and the cytoskeleton 33: 22-29.
186.
Yanai, M., Butler, J.P., Suzuki, T., Kanda, A., Kurachi, M., Tashiro, H., Sasaki, H. (1999) Intracellular
elasticity and viscosity in the body, leading, and trailing regions of locomoting neutrophils. American
journal of physiology 277: C432-C440.
187.
Yoshida, K., Inouye, K. (2001) Myosin II-dependent cylindrical protrusions induced by quinine in
Dictyostelium: anatgonizing effects of actin polymerisation at the leading edge. Journal of cell science
114: 2155-2165.
188.
Zhu, C., Skalak, R. (1988) A continuum model of protrusion of pseudopod in leukocytes. Biophysical
journal 54: 1115-1137.
189.
Zierold, K. (1991) Cryofixation methods for ion localisation in cells by electron probe microanalysis.
Journal of microscopy 161(2): 357-36.
61
Appendix
5. Appendix
APPENDIX 1
The cooling rate (Φ) is proportional to a function of the thickness of a sample (d) and the
thermal diffusion coefficient (κ):
Φ≈
κ
d2
Since the thickness behaves in an inverted squared relation to the cooling rate, a 100
times higher cooling rate will allow a 10 times increase of sample thickness.
The thermal diffusion coefficient (κ) is a combination of material specific coefficients:
κ=
K
ρ.Cp
with the thermal conductivity (K), the density (ρ) and the thermal capacity (Cp). None of
these coefficients can be manipulated by the experimenter to influence κ, and thereby
indirectly Φ. K, ρ, and Cp are dependent on the temperature as well, which makes
calculations and predictions much more complex.
APPENDIX 2
D-value (decimal reduction value) is the time needed to reduce a population to 10% of
it’s original population (or to destroy 90% of the population) e.g. by applying pressure.
A linear correlation is found in a semi-logarithmic plot (order of magnitude of cell
numbers against time). The modulator of the cell surviving rate are given by:
f(x)= -x/D + b
With D, the D-value and b the order of magnitude of the initial cell count.
62
List of publications
6. List of publications
PEER REVIEWED ARTICLES
•
Vanhecke, D., Graber, W., Herrmann, G., Al-Amoudi, A., Eggli, P., Studer, D.
(2003) A rapid microbiopsy system to improve the preservation of biological
samples prior to high-pressure freezing. Journal of microscopy 212(1): 3-12.
•
Claeys, M., Vanhecke, D., Couvreur, M., Tytgat, T., Coomans, A., Borgonie, G.
(2004) High pressure freezing and freeze substitution of gravid Caenorhabditis
elegans
(Nematoda:
Rhabditida)
for
transmission
electron
microscopy.
Nematology 6(3): 319-327.
BOOK CHAPTERS
•
Vanhecke, D., Eggli, P., Graber, W., Studer, D. (2005) A new microbiopsy system
enables rapid preparation of tissue for high pressure freezing. In Methods in
molecular biology (Taatjes DJ, Ed.). Humana press NY, USA. In press
IN PREPARATION
•
Vanhecke, D,. Eggli, P., Graber, W., Keller, H.U., Studer, D. (2005) Hitherto not
described inclusions in the front of Walker carcinosarcoma cells may play a role in
cellular motility.
PROCEEDINGS
•
Vanhecke, D., Claeys, M., Couvreur, M., Tytgat, T., Coomans, A., Borgonie, G.
(2001) Caenorhabditis elegans (Nematoda) as test subject for cryoimmobilisation.
In Proceedings of the 13th international C. elegans meeting, Los Angeles,
California, USA. p838.
•
Vanhecke, D., Graber, W., Herrmann, G., Al-Amoudi, A., Eggli, P., Studer, D.
(2003) Fast tissue preparation for cryofixation: a microbiopsy system for high
pressure freezing. In Proceedings of the 35th annual meeting of the Swiss societies
for experimental biology (USGEB/USSBE), Davos, Switzerland. p47.
•
Vanhecke, D., Eggli, P., Keller, H.U., Studer, D. (2004) Pseudovacuolar inclusions
at the front of Walker carcinosarcoma cells. In proceedings of the 13th European
microscopy congress, Antwerpen, Belgium.
•
Vanhecke, D., Eggli, P., Graber, W., Keller, H.U., Studer, D. (2005) Morphology of
frontal pseudovacuolar inclusions of Walker carcinosarcoma cells. In Molecules to
mind, USGEB/USSBE joint meeting 2005, Zürich, Switzerland. p77.
63
Curriculum vitae
7. Curriculum vitae
Dimitri Vanhecke
Nationality: Belgian
Date of Birth: September 30th 1978
Place of birth: Brugge, Belgium
Education:
•
1984-1990
Mariaschool
Zwevezele, Belgium
Primary school
•
1990-1996
St-Jozefsinstituut
Torhout, Belgium
Secondary school, diploma “doorstroming biotechniek”
•
1996-2001
University Gent
Gent, Belgium
Licentiate Biology (zoology), graduated with great distinction
•
2001-2005
University of Bern
Bern, Switzerland
PIAF PhD program on structural biology
64
Acknowledgements
8. Acknowledgements
Many thanks are going to my mentor, Prof. Dr. med. Peter Eggli, for letting me working
in the constructive environment of his lab, for fruitful discussions and creative
suggestions.
I’m grateful to Prof. Dr. Matthias Chiquet, my tutor, for giving me his opinion on ideas,
for showing me my flaws and errors and for keeping an eye on me.
Dr. sc. nat. ETH Daniel Studer is the person who taught me right from wrong, vitreous
from crystalline, tips and tricks and a lot more. Dani, I would like to thank you for
believing in me, and for offering me this chance.
Thankfulness is going to Prof. Dr. Hansuli Keller, for introducing me in the world of
blebbing cells. Thank you for the scientific contribution and the constructive discussions.
More thanks are going to the members of our lab who helped me, who were interested in
what I was doing and who fed me with ideas. Werner Graber, Dr. med. Gudrun Herrmann
and PD Dr. med. Alessandra Scotti: thanks a lot!
But my appreciation does not stay within the walls of our lab. Many thanks are going to
Dr. sc. Nat. ETH Barbara Rothen-Rutishauser for excellent help and tuning concerning my
light scanning thank microscopic problems, for discussions and ideas. To Dr. rer. nat.
Oliver Baum I would like to express my thanks for mental support, for his help with
experiments and scientific input. My appreciation goes to all the other members of the
Institute for Anatomy in Bern: Professors, assistants, technicians, secretaries and all the
other employees, who somehow/somewhere helped me in achieving my goal.
There are a lot of people outside of university I met and who spiced up life: Kristien, Jan,
Miguel, the entire 1. mannschaft of FC. Länggasse, Kathrine, Adi, Aldo, Jöre, Carlos,
Dania, the king of Bümpliz, Matthias and Nathalie: many many thanks. Also the old
group did not forget about me (far away from home). Ne welgemjinde mersi: Andy, Dirk,
Wiem, Kristof, Tom and Wouter!
Mom and Dad: great job! First because I’m here and second because I can be here.
Thanx to my brothers Mathias en Michiel: without both of you I would have collapsed.
65