The Dynamic Cell 34
Locomotion
S.K. Maciver Jan '03
Cell Locomotion
Cells move for a variety of reasons. Bacteria swim by beating their flagella in order to exploit newly created microenvironments and amoeba crawl to gather bacteria to feed on. The cells of vertebrates must move to heal wounds, fend
off invaders and the eukaryotic flagella is used to propel sperm cells toward eggs.
Cell type
Bacteria
Ciliate
Amoeba proteus
Neutrophil
Fibroblast
Scale speed*
10x length sec
100
0.006
0.01
0.0002
Speed (µm/sec)
10
1,000
3
0.1
0.01
Table 3 Speed record of different types of cell locomotion. *Scale speed expressed as the body lengths that the
organism covers every second (not really serious).
The Prokaryotic Flagellum
Bacteria invented the wheel! The bacterial flagellum is a helical
structure that drives the cell through the media like a propeller.
Hook
flagellin
Universal joint
The structure is rigid and turned by a rotatory motor at the base
where it connects to the bacteria's body. The rotary motor consist
L-ring
of several wheel-like discs one of which the M-ring (and/or
P-ring
possibly the S-ring) interact with the C-ring and studs to rotate the
whole structure. The rotary motor is very like a stepping motor!
The flagella is composed of a protein called flagellin which is
synthesized in the cell body and transported through the narrow
lumen of the growing flagella itself to polymerise at the tip as it is
about to exit the bacteria! This system has evolved into a syringe –
like mechanism to inject toxins into the cells of vertebrates during
infection (this is called "type 3 secretion"). There are two main
type of prokaryotic flagella, those belonging to gram positive (one
S-ring
Studs
membrane) and gram negative (two membranes) bacteria (Figure
M-ring
C-ring Figure 25
25). The bacterial flagellum is driven by a proton motive force
resulting from a gradient of protons. Bacterial chemotaxis is brought about by alterations in the direction that the motor
rotates in, this in turn is controlled by phosphorylation.
The Eukaryotic Flagellum
Although at first sight the flagella of eukaryotes is similar to the flagella of prokaryotes, our flagella are completely
dissimilar in structure, function and in the genes that encode their components. The principle component of the
eukaryotic flagella is the microtubule, a tubular array of
Outer
proteins of the tubulin family. Instead of rotating as the
Outer arm
doublet
prokaryotic flagella does, the eukaryotic flagella produces
contortions in shape that travel around the structure like a
Mexican wave. The term cilia is generally used to
describe small grouped structures less than 10µm, and
membrane
Inner arm
flagella tend to be single structures about 40µm. Our
cilia are considered to be a cellular organelle and are
Nexin
almost certain to be derived from a primitive protist cell
bridge
in the distant past. In the human body they are used in
Central
mucus membranes to driven mucus around (out of the
doublet
lungs), to drive sperm cells, but bizarrely, in development
Central
a single cilium is responsible for setting up the
Radial link
sheath
asymmetry of our internal organs (heart slightly to the left
head
etc.) rare mutations in the genes encoding this structure
Radial
cause situs inversus. Like many small things, the
link
Figure 26
eukaryotic flagella was first seen by Anton van
Leuwenhoek. The outer doublets are composed of microtubules and the outer and inner arms are dynein. Dynein is a
motor protein that works with microtubules much like myosin works on actin so that the whole flagellum is sent into
spiral motions as each set of arms (dynein) walks up the microtubules.
13
The Dynamic Cell 34
Locomotion
S.K. Maciver Jan '03
Crawling locomotion of cells.
It has been suggested that the primary driving force behind the evolution of the actin cytoskeleton was to permit the
equal division of components at cytokinesis. If this is so, then a close second must have been the ability to move! The
subject of cell locomotion has been a very long one. As far back as primitive microscopes were available scientists
have been studying the so called "Giant amoeba" (Amoeba proteus & Chaos carolinensis). At 1mm in length, these
cells are enormous and so were ideal subject for the early studies. The fact that these cells moved in "real time" (310µM/sec) meant that no time lapse photography was necessary either. The large size of these amoebae made it
possible to perform "Micrurgy", such as enucleation and tactile stimulation. It was concluded (Jennings, 1906;
Goldacre 1952) from such tactile studies that prodding the front of the cell caused the cell to change direction, whereas
a gentle prod in the rear made the cell accelerate transiently (as it would any sensible creature!). Goldacre (1953) found
that the uroid was contractile and that the cell could move even after the nucleus was removed with fine glass needle.
The finding that the uroid was contractile was the subject of about a hundred years of controversy as others (Allen,
1961) suggested that it was the front end of the cell that was contractile and active.
Sol-Endoplasm
Hyaline
Pseudopod
Gel- Ectoplasm
uroid
N
Released
adhesion
Glass slide
Adhesion
Endoplasm-ectoplasm transformation
A
Endoplasm to Ectoplasm transformation
Ectoplasm to Endoplasm transformation
membrane
B
C
Figure 27
Observations on the amoeba revealed that there were two convertible states of cytoplasm, endoplasm and ectoplasm.
Endoplasm (seen at the cell centre, thus the name) was fluid, while ectoplasm under the cell membrane is gellated and
comparatively static. During active locomotion, endoplasm flows forwards faster than the speed of the cell. As the
fluid endoplasm reaches the "hyaloplasm", a special optically clear form of ectoplasm, the flow diverted toward the
membrane whereupon the endoplasm gelled to form ectoplasm. Vesicles, crystals, and other visible cytoplasmic
inclusions are seen to become suddenly immobile having previously been seen to vibrate in Browning motion.
These transformations also take place in other cell types but are less visible because of the much smaller scale
and because they take place over a much longer time scale. However these transformations are quite clearly visible in
small amoeba such as Acanthamoeba, Dictyostelium and Naegleria, and also in highly motile human cells such as
macrophages and neutrophils.
The Frontal Contraction theory
Forwarded by Bob Allen (Allen, 1961) on the basis of the behaviour of cytoplasm released (accidentally in the first
instance) into media of various formulations. Allen found the liberated cytoplasm had some very peculiar properties,
the cytoplasm was contractile and isolated pools were seen to writhe producing squirting motions. Allen compared
these movements with the reversible gel to sol conversions that were clearly visible in the living cell. He proposed that
as the endoplasm to ectoplasm transformation took place, the forming ectoplasm contracted pulling the cell forward
(Figure 27B).
The generalised contraction theory
The generalised contraction theory eventually won the day and is more in favour of the rear contraction hypothesis
rather than the frontal contraction hypothesis. This hypothesis is only really applicable in its strictest sense to the giant
amoeba as they differ from other cell types in that the cortex is not attached to the plasma-membrane as it is in smaller
amoebae (Dictyostelium, Acanthamoeba, Naegleria) and vertebrate cells. However, a loose interpretation of the
generalised contraction theory is appropriate for some vertebrate cells at least, as there is a strong hydraulic component
14
The Dynamic Cell 34
Locomotion
S.K. Maciver Jan '03
to the locomotion of many cell types. The generalised contraction theory suggests that contraction occurs as the
ectoplasm transforms to endoplasm or in the more modern parlance, as gel disassembled to sol. Molecular mechanism
have been suggested for this transformation and a body of experimental evidence supports this (Janson & Taylor 1993).
Two types of crawling, lammelapodal spread and hydraulic contraction.
Many cell types in vertebrates adopt a very broad veil-like lamella or pseudopod at the cell front as they advance
forward by crawling. This morphology is best illustrated by the keratinocyte where most of the projected surface area is
occupied by the lamella (figure 28). This cell morphology does not have an obvious hydraulic component. The so
called "limax" amoeba (called that because they look like slugs) are totally hydraulic with no suggestion of
lammelopodal spread. The neutrophil is intermediate between these two extremes and displays lammelopodal spread
and a hydraulic contraction component.
Keratinocyte
“Limax” amoeba
Neutrophil
Figure 28 The keratocyte
The Myosins – Key motor proteins in Cell Motility and Locomotion.
The myosins are a group of motor proteins capable of transforming chemical energy in the form of ATP to movement
via the amplification (by levers) of conformational changes within the ATP hydrolysing head group. The myosins were
discussed in detail in Dr Paul McLaughlin's lecture (lecture number 17 on the 13th Nov'03) so the mechanisms of the
myosin motor itself will not be discussed here but rather the consequences of this action on cell locomotion. Although
there are a huge number of myosin family members we will be discussing myosin II, this is the two-headed myosin that
is the major myosin in muscle and is responsible for contractile functions (such as cytokinesis) in the vast majority of
non-muscle cells.
Head
Neck
p
ELC
RLC
p
Tail
Figure 29. Myosin II. Each catalytic head group is
controlled by two light chains, a regulatory and an
essential light chain. Light chains are calmodulin-like
proteins and wrap around a helical neck region. The
heavy chain tail regions wrap round each other too.
Each myosin II molecule self associates in an antiparallel manner regulated by phosphorylation at the
C-terminus (P).
Myosin II performs many functions in cells beside cell locomotion but probably its most important function is to
constrict the waist of the cell and so allow it to divide. In addition to be regulated by phosphorylation of the light
chains, the heavy chain is phosphorylated at the C-terminus (at the tail of the myosin molecule). Phosphorylation here
regulates the assembly of the myosin minifilament, small bipolar aggregations of myosins that allow the mini-filament
to exert a pulling force on actin filaments. The assembly of myosin minifilaments in cells is crucial to myosins
contractile function and is regulated by a large number of different types of kinases that are in turn activated by a large
number of signalling pathways to regulate their assembly.
PKC
Figure 30 A. sequential aggregation of
non-muscle myosins into mini-filament
(Sinard et al, 1989a)
B.
Myosin light chain kinase
phosphorylates the light chain on threonine
18 and serine 19 inducing a conformational
change from a folded shape, to an extended
active shape that is able to form
minifilaments. Phosphorylation at serine 9
by protein kinase C inactivates the myosin
even if it was previously activated by
MLCK, furthermore PKC phosphorylation
reduces the rate that the myosin can be
phosphorylated by MLCK.
A
15
Ser9
inactive
inactive
MLCK
PKC
Thr18
Ser 19
Active
B
inactive
The Dynamic Cell 34
Locomotion
S.K. Maciver Jan '03
Several myosin heavy chain kinases have been identified (table 4). In Dictyostelium there are three related enzymes
MHCK A, B and C (Liang et al, 2002). The latter, MHCK C seems to be involved primarily in cytokinesis while forms
A and B were loaclised with myosin II at the rear cortical region of moving cells, moreover their distribution here was
independent of myosin II as the same pattern was observed in the absence of myosin II. Dictyostelium also expresses
another MHCK that is related to protein kinase C (Rabin & Ravid, 2002), and like PKC in vertebrate cells this kinase
(MHCK-PKC) inhibits the formation of myosin filament assembly. MHCK-PKC is localised at the anterior of the
locomoting amoebae rather than the rear where many of the other kinases are.
Myosin II filaments are found throughout the cells of the fibroblastic type where they perform contractile functions
unrelated to cell locomo tion, but in cell types where the primary function of myosins is in cell locomotion, myosin
filaments are located at the rear of the cell. This is particularly true for hydraulic cells such as neutrophils and
Dictyostelium.
Figure 31 Myosin II is located at
Leading edge
Myosin II
Uroid
Keratinocyte
Neutrophil
Amoeba
the rear of the lammela in
fibroblasts (Kolega & Taylor,
1993) keratinocytes (Svitkina et
al, 1997) and neutrophils (Eddy et
al, 2000) and at the uroid of
locomoting Dictyostelium amoeba
(Yumura & Fukui, 1985), in
Acanthamoeba (Yonemura &
Pollard, 1992) neutrophils (Eddy
et al, 2000). Myosin II is also
concentrated at the uroid of
fibroblasts (Kolega & Taylor,
1993).
Myosin squeezes cytoplasm forward pulls the lamella forward, and rips off redundant adhesion at the rear.
Myosin II seems to perform three simultaneous functions in cells, the relative contribution of myosin to these three
areas dependent on the particular locomotory morphology of the cell. In the lamella, myosin II becomes progressively
organised in minifilaments arranged perpendicular to the direction of locomotion, toward the rear of the lamella the
highest concentration of mini-filaments often corresponds to the boundary where the lamella meets the cell body. A
series of experiments has been reported where cells are settled onto deformable surfaces onto which small beads have
been stuck to act as position markers. As cells translocate on these surfaces the pattern of force generation can be seen.
The forces generated are highly co relatable with the myosin II mini-filament distribution seen in the various cell types
(except in fibroblasts whose primary function is to contract and not to locomote). It has been possible now to make
Direction of cell travel
Figure 32 Cells locomoting on normal
substrates develop adhesions through
which the cell develops force. The
behaviour of the beads on the deformable
surface (lower figure). Wrinkles develop
between adhesions where contraction
occurs. These wrinkles pull the beads on
the latex surface towards the cell, or in
the case of expansion the advancing cell
pushes the beads away.
Beads
substrates that are so deformable that even the very weak forces that Dictyostelium exerts can be detected and studied
(Uchida et al, 2002). The contribution of myosin II to these forces has been assessed by comparing wild-type
Dictyostelium with a strain that lacks myosin II. The anterior region produced a pushing force as the cell retracted the
posterior and a pulling forces was detected in the rear. In line with the generalised contraction hypothesis the anterior
pushing force was interpreted as being generated by myosin II squeezing the cell hydraulically.
The experimental evidence for the roles of myosin in cell locomotion.
Since the discovery of myosin in non-muscle cells , most hypothesis on the problem of cell locomotion have been based
on the premise that somehow myosin and actin contractility was involved. It was therefore quite a shock when the
single myosin II gene of Dictyostelium was knocked out and that this did not completely prevent cells from locomoting
(De Lozanne & Spudich, 1987), it did however significantly slow them down and prevented the cells displaying a
chemotactic response. The explanation for this is that Dictyostelium only adheres weakly to the substrate and it has
many other myosins through which it can generate contraction (Dai et al, 1999). Inhibitory antibodies to myosin II
16
The Dynamic Cell 34
Locomotion
S.K. Maciver Jan '03
have been microinjected into adherent Acanthamoeba (Sinard & Pollard 1989b), like the situation with Dictyostelium,
the removal of myosin II function slowed but did not stop the cells from moving. Myosin II function has been reduced
too in vertebrate cells but this produced a more radical pattern of disruption (Honer et al, 1988). The locomotory rate
was reduced to almost zero and the cells seemed incapable of removing rear adhesions resulting in bizarre
morphologies. It seems that these fibroblasts were more affected that either Dictyostelium or Acanthamoeba since these
cells make much tighter adhesions. Neutrophils cannot be easily microinjected but instead the contribution of myosin II
to locomotion in these cells has been studied by the treatment of neutrophils with a myosin II inhibitor drug (BDM).
Figure 33 Myosin II minifilaments induce
contractile tension between adhesions. As
the cell progresses the older focal contacts
gather at the rear of the cell. A
combination of focal contact/adhesion
disassembly and a build up of strain results
in the release of the adhesion and the
elastic recoil of the material toward the cell
body. These recoil evens are often seen to
correspond to episodes of increased
protrusion at the leading edge
Forming Focal Contact
Mature Focal Contact
Disassembling Focal Contact
A
B
Cortical tube models for cell locomotion
If contraction/solation of the cortical tube takes place only at the uroid, then the shape of the cell would be
approximately cylindrical. In this most simple of cases a 10% reduction in the volume of the uroid would result in a
10% increase in volume at the anterior, since the diameter at each end is equal the width of the cell does not effect this
calculation. However, most cells are not cylindrical but are variously tapered toward the uroid. The shape of many
hydraulic cells ectoplasmic cortex is approximately a semi-ellipsoid (Figure 34a). Sequential contraction of the cortex
by myosin II coupled with solation results in the formation of sol which can then be recycled to new ectoplasmic cortex
at the front of the cell. Obviously there is a relationship between contraction and new cortex formation and this
determines the speed of locomotion. If we compute the length of cortical portions created by the same contraction of
cortexes with arbitrary units (Figure 34b) it can be seen that the width of the cell is not important in determining the
speed but rather it is the length of the cell cortex. This in fact is supported by the literature on amoeba of various
lengths and thicknesses.
Contraction
uroid
Radius
Cell length
100
150
200
20
40
80
0.67
0.67
0.67
1.3
1.3
1.3
1
1
1
B. In the above example (Figure 34b) the
relationship between the number of cortical portions
produced by each 10% reduction by contraction of
the cortex depends solely on the length and not the
cell width. For a length of 150 units, 1 unit of new
cortex is produced by each 10% reduction in volume
A
Formation of new cortex
Volume = 2π x half the(length.Width.Height)
3
Figure 34
Signal Transduction and Cell Locomotion
As one might expect, the signal pathways that regulate cell locomotion overlap to a large degree with those activated
during the chemotactic response. Activated Rac binds and activates Ca 2+ and calmodulin dependent kinase which can
then phosphorylated Myosin II tails causing disassembly. Stimulation of both the PAK1 and PI(3)kinase pathways
results in AKT/PKB phosphorylation and activation through p38MAPK and directly by PI(3)kinase. AKT/PKB binds
myosin when activates (Tanaka et al, 1999) but the consequence of this is not yet clear. Activation of MK2 results in
the phosphorylation of myosin light chain and activation of the ATPase. The simultaneous release of the heat shock
protein HSP27 may also affect actin polymerization since this protein (under poorly understood conditions) binds actin.
A possible negative feed back mechanism may operate through PAK1 phosphorylation of MLCK which inhibits its
17
The Dynamic Cell 34
Locomotion
Ca 2+/Cam Kinase
RAC
PAK1
CDC42
PI(3)K
P
RAS
p38
PIP2 PI(3,4,5)P
3
MK2
Hsp27
MK2
Hsp27
S.K. Maciver Jan '03
activity. MLCK is activated by the G-protein
Rho and Rho also activated Rho-kinase which
activates myosin by direct phosphorylation of
the light chains. The details of how these
interconnected pathways manage to work
together to determine when and where myosin
filaments are formed and when and where
they are activated are not at all clear, but in
the neutrophil at least calcium seems to have a
major effect in myosin activation (Eddy et al,
2000).
AKT
PKB
Rho
MLCK
AKT
PKB
Rho kinase
Figure 35 Connecting myosin II activation to
the major signalling pathways in neutrophils
and Dictyostelium.
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De Lozanne, A. & Spudich, J. A. (1987) Disruption of the Dictyostelium myosin heavy chain gene by homologous
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Queries to S. Maciver Room 444, Hugh Robson Building, George Square. E-mail [email protected].
Fax/Tel 0131 650 3714.
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