Bioscience Reports 4, 451-466 (1984)
Printed in Great Britain
Microinjection
red-ceil-mediated
#51
of c u l t u r e d c e l l s using
fusion and o s m o t i c lysis of p i n o s o m e s :
A r e v i e w of m e t h o d s and a p p l i c a t i o n s
Review
Mary Ann McELLIGOTT and 3. Fred DICE
Department of Physiology and Biophysics,
Harvard Medical School, Boston, MA 02115, U.S.A.
Proteins and other macromolecules can be injected into
cultured ceils by several different methods.
Here we
review the strengths and limitations of two of these
methods, red-celJ-mediated microinjection and osmotic:
lysis of pinosomes, and indicate how they may be
successfully applied to the study of cultured cells.
Introduction
Powerful new techniques have been developed which enable
scientists to introduce macromolecules into the cytosol of cultured
ceils.
Among the methods currently in use are microneedle injection
( I - 3 ) , red-cell-mediated microinjection (3-6) 9 injection by liposome
fusion (7)8)) and injection by osmotic lysis of pinocytic vesicles (9).
M i c r o i n j e c t i o n by microneedle gives the advantage of direct,
efficient delivery of large amounts of almost any macromolecule into
the cytosol (1,2) I nuclei (10,11), or even mitochondria ( I 0 ) of
recipient cells.
The major Jimitation of this approach is that only
about 500 cells can be microinjected per hour, or about 0.03% of the
number of fibroblasts on a 100-ram culture dish.
Therefore) biochemical studies requiring large numbers of microinjected cells are not
practical.
Delivery of macromolecules to the cell using liposomes is attractive
in that large macromoiecuJes (7,8) and even whole viruses (12) can
be encapsulated into large unilamellar vesicles. The major drawbacks
of liposome-mediated microinjection are that certain liposomes fuse
poorly (13) and many liposomes strongly adsorb to cell membranes
(8,12,13) w i t h o u t actually fusing.
Since adsorbed liposomes may
eventually fuse with the plasma membrane or may be taken up by
endocytosis (7,8)12,13), the use of liposomes to inject proteins is
currently restricted to studies in which variability in the method of
entry and the presence of unfused vesicles do not complicate the
interpretation of results.
The techniques of r e d - c e l l - m e d i a t e d microinjection (4.-6) and
osmotic Iysis of pinosomes~ (9) have been extensively used in our
laboratory to study mechanisms of intracellular protein degradation
(14-17).
Both of these procedures permit simultaneous injection of
9 1984 The Biochemical .Society
452
McELLIGOTT & DICE
the majority of ceils on a tissue-culture plate, and one person can
easily microinject [0 plates of cells in 5 minutes.
Here we briefly
r e v i e w how t h e s e t e c h n i q u e s ha ve been used~ the strengths and
limitations of each method, and the areas of cell biology in which
these approaches might be especially useful.
Procedures
Red-cell-mediated microinjection
The technique using red ceils as 'minisyringes' for microinjection
was introduced about ten years ago (4-6).
Red ceils are loaded with
macromolecules by either preswell hypotonic lysis followed by resealing
(18-20) or by dialysis loading (6,21).
The loaded red cells are then
fused to the tissue-culture cells using either inactivated Sendai virus
( 4 - 6 ) or the chemical fusogen polyethylene glycol (PEG) (14,22).
The ex act protocols for optimal fusion vary somewhat for di fferent
c e l l t y p e s and in different laboratories, but typical protocols are
described below.
Sendai virus
The fusion mixture for microinjection using Sendai virus (5,6,23)
consists of cultured ceils in suspension, a hundred times the number of
l o a d e d r e d - c e l l ' g h o s t s ' compared to cultured cells, and 400-1600
hemagglutinating units of u.v.-inactivated Sendal virus in 12g mM KCI,
32 mM NaCI, and 20 mM Tris buffer at pH 7.4. The mixture is kept
on ice for 10-15 min and then incubated with shaking for 20-30 min at
37~
Cold Tris-saline buffer is added to t e r m i n a t e fusion and the
ceils are washed in culture medium several times before plating.
The exact mechanisms of fusion by Sendal virus are still being
clarified but are believed to involve the following steps (3,24):
The
Sendal virus first integrates into the cell membrane, and two viralm e m b r a n e p r o t e i n s a r e r e q u i r e d for fusion.
One protein, which
possesses the agglutinating and neuraminidase activity, interacts with
the sialic acid residues of sialoglycoproteins and sialoglycolipids in the
plasma membrane of the adjacent cell.
The neuraminidase probably
a c t s to l ow e r e l e c t r o s t a t i c repulsion between the membranes by
c l e a v i n g t h e highly charged sialic acid moieties on the membrane
surface.
The second protein promotes the actual membrane fusion by
stimulating aggregation of intramembrane proteins, thereby exposing
l i p i d - r i c h a r e a s of the bilayer, where fusion is thought to occur.
Recently, fusion of loaded ghosts with fibroblasts was accomplished
using only these two purified Sendai-virus proteins (25) as the fusogen.
R e c o n s t i t u t e d v i r a l - m e m b r a n e e n v e l o p e s have also been used as
vehicles to introduce macromolecules into ceils (26,27).
Polyethylene glycol (PEG)
PEG is most commonly used as a fusogen with ceils in monolayer
cultures (Fig. l) (14,17,22). The cultures are first rinsed with Hanks'
balanced salt solution to remove serum proteins which are thought to
i n t e r f e r e with fusion. The solution is then removed from the culture,
and i00-150 loaded red-cell ghosts per cultured cell are added to the
monolayer in a small volume of Hanks' and distributed over the plate.
MICROIN3ECTION
OF
CULTURED
CELLS
#53
PEG-1000 (40%, diluted in culture medium) at 37 ~ is added to the
plate and a f t e r 1 min is diluted with #.5 ml of culture medium. The
cultures are then rinsed 3 times with 10 ml of Hanks' containing 596
f e t a l bovine serum to remove non-fused ghosts.
The mechanism of PEG-mediated fusion appears to be similar in
some ways to that induced by Sendai virus. PEG has a high capacity
to bind water (28~29), which may destabilize the membrane (28,30)
and p r o m o t e the aggregation of intramembranous particles (28~31)~
thereby exposing lipid-rich areas of the membrane. A combination of
d e h y d r a t i o n and d e s t a b i l i z a t i o n of t h e s u r f a c e potential of the
m e m b r a n e ( 28) p r o b a b l y allows t h e c l o s e c o n t a c t between cell
membranes which is a prerequisite for fusion. Several reports suggest
that PEG alone is not adequate for fusion since the fusogenic capaci t y
of PEG is lost or diminished (2g~32~33) upon purification by et her
extraction.
The crucial contaminating agents in PEG are believed to
be an antioxidant and/or a polymerization c a t a l y s t (28132). However~
purification of PEG may not diminish the fusogenic capaci t y of all
brands of PEG (33). It is possible that levels of contaminants in PEG
may be especially critical for fusion of red ceils to some cell types
but not to o t h e r s .
In any event~ the well known variability in
fusogenic potential of commercially available PEG may be explained if
the fusogenic action requires both PEG and c o n t a m i n a n t s .
In our
experienc% this variability applies to PEG from d i f f e r e n t manufact u r e r s as well as to d i f f e r e n t p r o d u c t i o n Jots f r o m the same
manufacturer.
,Hb~Hb Hb
~
~
~/3HANKS ~ e
1.1._ysi_ss
SALTS
2. Loadinq_ ,.*PROTEIN
PROTEIN** * ~~1 *
Hb
~6~
2,
hb
Y
H
* * . ~_~
PROTEIN.~
l
Hb
9* * .
9
* .
#
"~ [C~~
(*,)
'3.~Rese
OIVOL
*
.='~*PROTEIN
*
, /
lOXH,4NKS'~
~
57 ~,lhr
o,io _
POLYETHYLENE GLYCOL
40 %, Imln ~
4. Fusion
-
--
MYOTUBES
-..~';:,..~::)~Z~. ~ i ! :,
',' :~ ~:T ~'! .~!~
!F~~:"~'
~:
!,:? : .
FIBROBLASTS
Fig. I.
Red-cell-mediated microinjection.
#5#
McELLIGOTT & DICE
Osmotic lysis of pinosomes
The most recent procedure for introducing macromolecules into the
cytosol of cultured cells (Fig. 2) is based on the pinocytic uptake of
culture medium containing the protein of interest, 0.5 M sucrose, and
1096 PEG-1000, followed by osmotic lysis of the pinocytic vesicles (9).
Fluid phase uptake in 10 rain is approx. 0.1 pl per 2 x 10 s cells for
fibroblasts (9).
For studies of protein degradation in human fibroblasts, we routinely add 5 x l0 T c.p.m, of radioactive protein in one
ml of medium so that 5000 c.p.m, are introduced per plate. After 10
rain of pinocytosis the cells are washed with 60% culture medium/#0%
water.
This hypotonic wash lyses the hypertonic pinocytic vesicles,
and the pinocytosed macromolecules are released into the cytoplasm.
The role of the PEG-1000 is not certain but may serve to destabilize
the pinosome membranes (9).
Successful Applications of Microinjection
R e d - c e l l - m e d i a t e d microinjection has been successfully used to
inject many different proteins (see ref. 3 for review) into several
d i f f e r e n t cell types (Table 1).
Osmotic lysis of pinosomes is a
relatively new technique that has not been extensively evaluated, but
should be useful for similar studies.
Prote/n(*J
0 5 M Sucrose
'0% Po/#p~eneG/yco/
2. LYSISOF PINOSOMES
40% D M ~ . , ~ , , ~
I.PINOCYTIC UPTAK______EE
.... '.~" *
Z, WASH
Fig. 2.
Osmotic lysis of pinosomes.
MICROIN3ECTION
OF
CULTURED
CELLS
455
Table I. Microinjection of proteins into different cell types
Cell type
Fusogen and reference
Carcinoma:
human
rat mammary
PEG(62)
SV(51)
Chick muscle
PEG(17)
Ab, BSA, Hb, RNase A
Ehrlich ascites tumor cell
SV(46,71)
Ab, nonhistone chromosomal proteins
SV(42,52)
PEG(28,55)
SV(38,48)
PEG(14-16,28,35,50)
OLP(9)
PEG(56)
PEG(22,28,56,61,64)
SV(39,46,57)
HMg-I, HMG-2
HGPRT
Hb
Ab, BSA, lysozyme, ovalbumin, RNase
A, RNase S, RNase S-protein, RNase
S-peptide, polyglutamate:tyrosine
(i:I), ubiquitin
arginase, Hb
Ab, BSA
Ab, aidolase, arginase, BSA, carbonic
anhydrase, citrate synthase,
cytochrome o, ferritin, Hb, HMG-I,
NMG-2, HMG-T, LDH, lysozyme,
myoglobin, PK, RNase A, STI, TK
Ab, HRP
Ab
Ab, BSA, SV40 and SVSO T antigen
Ab, BSA, HMG-T, myoglobin, ferritin
Friend erythroleukemic cells
SV(40,71)
Ab
HeLA
PEG(53,54,63)
SV(34,36,37,39,
42,44,47,52,76,84)
BSA
Ab and fragments, adenylate kinase,
aldolase, BSA, carbonic anhydrase B,
carboxypeptidase A, citrate synthase,
oytochrome c, slastase, ferritin, Hb,
HMG-I, HMG-2, LDR, lysozyme,
myoglobin, ovalbumin, phospholipase
A2, PK, RNase A, ST!, ubiquitin
ubiquitin
Fibroblasts:
bovine
hamster
human
sv(25,3g)
PEG(28,56,75)
SV(5,9,23,25,27,34,
37-39,46,52,57)
rat
monkey
OLP(44)
Hepatocytes:
rat
PEG(22,58,72)
Proteins microinjected
Ab
SV(6,37,39,43,45,
49,72,7])
BSA, catalytic suhunit of protein
kinase, HRP
Ab, aldolase, BSA, ferritin, ?[P~P,
LDH, lysozyme, PK, ubiquitin
Human amnion cell
SV(4,46,57,71)
Ab, Hb
Lymphocytes:
human
murine
SV(65)
PEG(59,60,75)
B-cell cytoplasmic proteins
Ab, BSA, Hb
Mouse adrenal tumor
SV(39)
BSA
Mouse melanoma
SV(37)
Ab, aldolase, BSA, LDH,lysozyme, PK
Mouse myeloma
PEG(75)
BSA
Mouse monocyte
PEG(75)
BSA
Mouse neuroblastoma
SV(37)
Ab, aldolase, BSA, LD~, PK
Abbreviations: Ab, antibodies; BSA, bovine serum albumin; Hb, hemoglobin; HGPRT,
hypoxanthine guanine phosphoribosyl transferase; ~G-I,2, high-mobility-group proteins;
HMG-T, high-mobility-group proteins from trout; HRP, horseradish peroxidase; LDH, lactate
dehydrogenase; OLP, osmotic lysis of pinosomes; PEG, polyethylene glycol; PK, pyruvate
kinase; RNase A, ribonuclease A; STI, soybean trypsin inhibitor; SV, Sendal virus; TK,
~hymidine kinase.
456
McELLIGOTT & DICE
Intracellular protein degradation
Microinjection has permitted the analysis of the mechanisms and
regulation of protein catabolism at a level of detail that was not
previously possible.
In fac% the majority of reports using microi n j e c t i o n to d a t e have focused on this area of research (34-49).
M o d i f i c a t i o n of s e v e r a l well characterized proteins, including IgG
(36,37), hemoglobin (38), and RNase A (16), prior to microinjection
has permitted the analysis of the influence of protein structure on
half-life.
Microinjection has provided the sole means of assessing the
r e l a t i v e c o n t r i b u t i o n s of lysosomal and non-lysosomal pathways of
degradation without the use of inhibitors (34,35).
In these studies
proteins were labeled with radioactive sucrose (34) or raffinose (35)
prior to microinjection, and the location of the sugar-lysine degradation p r o d u c t i n d i c a t e d t h e subcellular location of its catabolism.
Microinjection of specific proteins into cells maintained under defined
media conditions (16,47) or into cells of different ages (15) promises
to c l a r i f y
h o w r a t e s o~[ p r o t e i n d e g r a d a t i o n are r e g u l a t e d .
Microinjection also affords the opportunity to search for intermediates
in p r o t e i n d e g r a d a t i o n (36,38-40).
Microinjection techniques have
f u r t h e r been used to analyze membrane protein degradation (27)
following transfer of protein (incorporated into Sendai-virus envelopes)
into cell m e m b r a n e s , and to s t u d y degradation of mitochondrial
membranes after fusion with plasma membrane (41).
Intracellular compartmentalization of proteins
M i c r o i n j e c t i o n has been u s e f u l in s t u d y i n g t h e i n t r a c e l l u l a r
m o v e m e n t s and localization of certain cellular proteins.
Diffusion
c o e f f i c i e n t s determined for proteins microinjected into the cytosol
(50) suggested that the cytosol was viscous, probably due to organized
cytoskeleton.
Some m i c r o i n j e c t e d p r o t e i n s e n t e r the n u c l e u s
(36,42,51,52) while others are excluded (14,17,36,38).
This differe n t i a l d i s t r i b u t i o n of a c e r t a i n protein between the nucleus and
cytoplasm appears to depend on both the protein and the cell type
(42,52-54).
Microinjection studies have indicated that molecular size
(36,51) is one limiting factor in nuclear entry, but further analysis is
r e q u i r e d to determine the additional mechanisms of specificity for
import of proteins into the nucleus.
Intracellular function of proteins
Intracellular protein function has been analyzed by microinjecting
cells with the protein of interest or an antibody to that p r o t e i n .
Enzymes microinjected into ceils that are genetically deficient in the
enzyme can restore the wild-type function (5,25,55).
Furthermore,
microinjected antibodies can protect cells from subsequent exposure to
toxic antigens (9,46956,57).
Microinjection offers unique advantages
for the study of intracellular mediators of hormone action (58-60),
c e l l u l a r transformation (22,61-64), and cell signalling factors (65).
One study, for example, used microinjection to study the intracellular
m e s s e n g e r of T-cell-replacing factor (65).
Microinjection of the
cytosol from a B-lymphoblastoid cell line (CESS) stimulated by this
MICROIN3ECTION
Table 2.
OF
CULTURED
CELLS
#.57
Effect of radiolabeling on protein structure
Proteins were iodinated using lactoperoxidase and glucosoxidase
(14) and tritiated by reductive methylation (16).
Susceptibility
to proteolytic
attack
Protein
Isotope
Enzymatic
activity
RNase A
125I
3H
nc
nc
nc
Lysozyme
125I
3H
(-)
(-)
(+)
(+)
Aldolase
125I
3H
(-)
nc
Glyceraldehyde-3-phosphate
dehydrogenase
125I
3H
(-)
nc
Aspartate aminotransferase
3H
(-)
Bovine serum albumin
125I
(+)
Ovalbumin
125I
(-)
nc, no change; (-), decrease;
nc
(+), increase.
f a c t o r a c t i v a t e d non-stimulated cells to produce Igs.
In addition)
microinjection of the catalytic subunit of the cAMP-dependent protein
kinase directly demonstrated that this enzyme mediated cAMP action
(-58).
Potential Limitations
Although microinjection techniques are clearly of immense value)
there are certain limitations of which to be aware.
One potential
problem common to both r e d - c e l l - m e d i a t e d microinjection and osmotic
lysis of pinosomes is that the process of labeling proteins to high
specific radioactivities may alter protein structure.
Certain proteins
are denatured by iodination, as assayed by loss of enzyme activity and
a l t e r e d s u s c e p t i b i l i t y to proteases in vitro ([#, Table 2).
Some
e n z y m e s which a r e i n a c t i v a t e d by iodination may retain enzyme
activity after reductive methylation while others are inactivated by
either labeling procedure.
Therefore) for physiological relevance) any
study using labeled proteins should demonstrate that the protein retains
native properties after labeling.
E x p e r i m e n t s a t t e m p t i n g to measure the degradative rate of a
protein (39) may also be complicated by the fact that prosthetic
groups
such as the p o r p h y r i n ring of myglobin (39) or enzyme
cofactors may also be tagged by the labeling procedure.
Some cell types may possess dehalogenase (66) or demethylase
(67) activities which could cleave the radioactive iodine or methyl
group from the protein.
Therefore) for degradation experiments) it
should be established that the radioactivity released through degradation is in the form of iodotyrosine (68) or dimethyllysine (69).
$58
McELLIGOTT & DICE
Red-cell-mediated microinjection
Loading
Several limitations of this technique concern the use of red cells as
carriers for the macromolecule to be transferred. Some of these have
been reported previously (3~20). Very large or elongated proteins do
not load e f f i c i e n t l y since the diameter of the pores of the lysed
ghosts is only about 20-50 nm (3).
Molecular sieving limits the
loading of large macromolecules (3).
There is a strong negative
surface charge (20) on the red cells, so certain proteins with net
positive charge, such as histones (20) and lysozyme ( l # ) , tend to
adsorb to the red-cell membrane and reduce fusion efficiency.
one further l i m i t a t i o n of the loading procedure is that the uptake
of macromolecules to be loaded per ghost is limited to 35% (3), using
published procedures.
Therefore, it is advantageous to recover the
labeled p r o t e i n that is not loaded.
We have subjected the first
supernatant from ghosts loaded with 12sI- or 3H-labeled RNase A to
i s o e l e c t r i c focusing ( 7 0 ) .
The RNase A (pI 9.1) is completely
separated from hemoglobin (pI 7.1), and can be dialyzed to remove
a m p h o l y t e s ~ lyophilized~ and used again.
Such RNase A appears
equivalent to freshly prepared RNase A in terms of its degradation
after microinjection.
Whether other proteins can be repurified and
reused in subsequent experiments remains to be shown. Undoubtedly~
it will depend on whether the protein is easily denatured at 37 ~ or is
susceptible to oxidation in the presence of hemoglobin.
Proteolysis by red cells
Most proteins do not appear to be degraded by red-cell proteases
(14,16~39,71) during the loading procedure.
However~ some proteins
(RNase S peptide~ aldolase~ glyceraldehyde-3-phosphate dehydrogenase)
are appreciably hydrolyzed to smaller-molecular-weight products during
the 1-h incubation at 37~
to allow resealing of the ghosts (16;
Backer~ Netland~ & Dice~ unpublished data).
Such proteolysis can
probably be prevented with protease inhibitors~ but unless the inhibitors
can be quantitatively removed they will be introduced into recipient
cells during microinjection.
Red-cell lysis
There are also several potential problems associated with the fusion
step of microinjection whether Sendai virus or PEG is used as fusogen.
Several investigators (6,20,32,72,73) report that Sendal virus and PEG
lyses 20-60% of the loaded red cells. The cell cultures are therefore
exposed to free protein in the presence of fusogen.
'Mock microi n j e c t i o n ' studies in which free protein is applied to ceils in the
presence of PEG demonstrate that certain proteins can associate
extensively with the cell monolayer (15,16). We have estimated that
the amounts of free protein associated with the cells using PEG are
variable and can range from less than 0.1% to as much as 20% of the
applied protein.
The estimates of cell-associated protein for 'mock
microinjection' studies using Sendal virus range between 7% (00) and
25% (23) of the amount microinjected.
The mechanism of binding of free protein is not known but can
lead to serious complications when binding is extensive.
For example~
MICROIN3ECTION
OF
CULTURED
CELLS
459
our unpublished results indicate that 12sI- and 3H-aldolase adsorb to
ceil monolayers, and large amounts remain adsorbed for 4;~ hours.
Thus, inaccurately high fusion ratios (20 ghosts:tissue culture cell) and
long half-lives are calculated.
The adsorbed aldolase is eluted if
serum is present in the culture medium but less is eJuted in the
absence of serum.
Therefore, at the end of the experiment, cells in
the presence of serum appear to contain less radioactive protein than
those in the absence of serum. Of course, further problems can arise
if adsorbed protein is eventually endocytosed by the cells.
Effects of microinjection on recipient cells
The fusogens used for microinjection can affect cellular metabolism
in a variety of ways.
Cell death after Sendai-virus-mediatecl microinjection is of the order of 1096 (71-73) although surviving fibroblasts
and HeLa cells are capable of further divisions (20,71). The cloning
efficiency of HeLa cells is not altered by fusions using Senclai virus
( 2 0 ) , and Sendal-virus-mediated fusion does not affect rates of
endogenous protein breakdown in fibroblasts (38) or Friend erythroleukemic cells (40).
Certain Jots of PEG from some sources do not
appear to a f f e c t growth of fibroblasts at concentrations that are
fusogenic (1%15).
However, in one study, the cloning efficiency of
ceils after brief exposures to PEG was greatly inhibited (56). PEG at
4096 does not cause detectable fibroblast-fibroblast fusion (14). PEG
has been observed to accelerate rates of degradation of long-lived
proteins for the first 2 h after microinjection in fibroblasts (14), but
other studies using d i f f e r e n t lots of PEG showed no effect on
endogenous protein catabolism in fibroblasts (15).
Rates of protein
synthesis were not affected in fibroblasts (14,15) but were altered in
hepatoma ceils (58) for 8-12 h after microinjection using PEG and
phytohemagglutinin.
There is a Joss of approx. 7-2596 protein per
plate of cells following PEG-mediated microinjection of myotubes (17),
i n d i c a t i v e of some cell death.
However, the remaining rnyotubes
function normally (17) for as long as 2 days after microinjection,
based on rates of protein synthesis and degradation.
Removal of non-fused ghosts
Removal of non-fused ghosts after Sendai-virus microinjection is
accomplished by e x t e n s i v e washing by centrifugation followed by
replating.
Non-fused ghosts cannot be completely removed after
PEG-mediated microinjection but estimates with fibroblasts (14,15) and
myotubes (17) reveal that the number of ghosts left on the plate is
equivalent to less than 196 of the radioactive protein microinjected.
Cultures can be incubated with 0.8396 NH. CI for l0 rain after fusion
to insure lysis of remaining ghosts (23). ~ultured ceils do not appear
to be affected. This treatment might produce a transient decrease in
protein degradation unless the NH4CI can be thoroughly washed away.
Efficiency of fusion
The overall e f f i c i e n c y of m i c r o i n j e c t i o n is r a t h e r low, since only
1-3 ghosts fuse per t i s s u e - c u l t u r e cell in the p r e s e n c e of a 150-fold
e x c e s s of loaded ghosts.
H o w e v e r , c a l c u l a t e d fusion ratios are an
a v e r a g e for a p l a t e . In f a c t , some r e c i p i e n t ceils fuse with more than
one r e d - c e l l ghost, while o t h e r ceils r e m a i n u n i n j e c t e d .
Microscopic
460
McELLIGOTT & DICE
studies indicate that 25-90% of the cells in a culture dish are actually
microinjected (14,17,38739ff2).
The factors that make one cell more
receptive to fusion than another are not understood.
Efficiency of fusion can be increased when phytohemagglutinins are
used to aggregate the e r y t h r o c y t e s with the tissue-culture cells
(63~72).
However, phytohemagglutinins have been shown to have
insulin-like effects (74) which would preclude examining the effects of
c e r t a i n hormones after microinjection.
Also, the removal of the
non-fused ghosts is extremely difficult when phytohemagglutinins are
used (72).
A very high efficiency of microinjection is obtained (75) when
cells are treated with avidin-modified antibodies before PEG-mediated
fusion to biotin-coupled loaded ghosts.
Specific cell types can be
targeted depending on the antibody. This technique may be useful for
certain cell types, but removal of nonfused ghosts is an anticipated
problem.
The l i m i t e d fusion efficiencies could probably be enhanced by
detailed optimization of fusion protocols, although work in this area
has been limited.
Dr. 3. Berger in our laboratory has found that
fusion efficiency in IMR-90 fibroblasts can be enhanced 3-fold by
d i l u t i n g t h e P E G w i t h 0.3 M glucose instead of c u l t u r e medium
(unpublished r e s u l t s ) ,
E f f i c i e n t fusion of red ceils to r e c i p i e n t ceils
was also obtained by c e n t r i f u g i n g p h y t o h e m a g g l u t i n i n - t r e a t e d red cells
and H e L a ceils onto coverslips which w e r e then i n c u b a t e d with PEG
and DMSO ( 7 6 ) ,
Fusion e f f i c i e n c i e s may also be improved more
s y s t e m a t i c a l l y when we b e t t e r understand the m e c h a n i s m s of m e m brane fusion,
C r e a t i v e new ways to i n t r o d u c e molecules into ceils are also being
developed,
E l e c t r o f u s i o n of cells (77) is a promising new m e t h o d
which can fuse large numbers of ceils quickly and in a more c o n t r o l l e d
m a n n e r than c h e m i c a l or viral fusion,
Whether loaded ghosts can be
e f f e c t i v e l y fused with t i s s u e - c u l t u r e ceils using this t e c h n i q u e remains
to be established.
Unanswered questions
Several reports (37,38) have noted that some microinjected proteins
are degraded with complex kinetics. These kinetics could be due to a
heterogeneous population of labeled protein or to variations in the
cellular localization of the protein after microinjection, but these
p o t e n t i a l explanations remain to be verified.
Another perplexing
observation is that in addition to degradation products, undegraded or
only partially degraded protein is released from the cell in potentially
significant amounts (36,37).
Our unpublished observations show that
acid-precipitable protein released from IMR-90 fibroblasts in which
cellular protein is labeled with radioactive leucine for 2 days is less
than I0% of the acid-soluble radioactivity released.
In contrast,
release of acid-precipitable microinjected protein from cells is as high
as 50% of t o t a l radioactivity released9 and this release does not
appear to be due to cell death (36,37).
Rechsteiner suggests that
undegraded microinjected protein released into the medium may be due
to uptake of cytosol into membrane-bound vesicles which can then be
transported to sites of degradation or to the plasma membrane where
MICROINJECTION
OF
CULTURED
CELLS
they can be released from the cell (36,37).
thesis certainly merits further investigation.
461
This interesting hypo-
Osmotic lysis of pinosomes
Efficiency
L a r g e a m o u n t s of r a d i o a c t i v e p r o t e i n are r e q u i r e d for this
procedure since the fluid-phase volume endocytosed in l0 min is small
(0.05 ~1/t0 G fibroblasts) (9).
However, we have found that media
containing SH-RNase A can be frozen and reused at least 5 times.
The protein does not appear to be altered by this t r e a t m e n t and is
degraded at equivalent rates in subsequent experiments (Backer and
Dice, unpublished d a t a ) .
Moreover~ larger amounts of the protein of
interest can be introduced into the ceils by multiple rounds of uptake
and lysis (9).
Protein adsorption
R e l a t i v e l y few proteins are endocytosed entirely by fluid phase
(9,78).
Fluid-phase endocytosis can be verified by demonstrating that
the rate of uptake of the protein is the same as for a fluid-phase
marker, such as [l~C]sucrose.
If the rate of protein uptake exceeds
that of the fluid-phase marker, then the protein is entering the cell by
adsorptive as well as fluid-phase processes. We have found that both
z2sI-labeled and 3H-labeled RNase A enter the cell by fluid-phase
endocytosis.
H o w e v e r , RNase S-protein, S-peptide9 S-peptidez_z49
S-peptide~_]3 , aldolase9 G3PDH, lysozyme, IgG, and iodinated horseradish peroxidase are taken up at 3-10 times fluid-phase rates (Backer,
McElligott~ and Dice, unpublished d a t a ) , suggesting that this may be
the more common route of entry.
Complex degradative kinetics
We have measured the degradation of proteins introduced into the
cell by pinocytic lysis and have observed a very fast initial phase of
degradation, which is complete by l0 h~ followed by a Mower phase.
The fast phase can account for as much as 60-9096 of the introduced
protein9 and may represent the degradation of the proteins which
entered by adsorptive pathways.
In support of this hypothesis, the
a m o u n t of p r o t e i n degraded in the rapid initial phase is directly
p r o p o r t i o n a l to the a m o u n t of protein that entered the cell by
adsorptive endocytosis.
Furthermore, lysis of pinosomes may be
i n c o m p l e t e since immediately after exposure of cells to hypotonic
medium, Percoll gradients reveal that more than 5096 of the pinocytosed 3H-RNase A is either already associated with lysosomes or
still contained in pinocytic vesicles (McElligott and Dice, unpublished
observations).
The remainder of the radioactive protein does appear
go be released into the cytosol by the osmotic lysis. The slow phase
of degradation may correspond to the breakdown of this free componen% since it is only this phase whose rate is accelerated by serum
withdrawal.
By waiting 10-12 h to start degradation measurements,
the catabolism of the fraction that is released into the cytosol at the
time of lysis can be studied. Rapid transfer of endocytosed material
to l y s o s o m e s has been observed by others (78-80).
Delivery to
l y s o s o m e s of r e c e p t o r - m e d i a t e d endocytosed materials (79) begins
t~62
McELLIGOTT & DICE
within 5 rain of uptake and appears to be complete by 30-60 rain in
most cells (78). Fluid-phase pinocytosed HRP also begins appearing in
lysosomes within 10 rain (80).
For each cell type, an optimum time
of pinocytosis should be established, since variations may exist in the
rate of transfer of endocytosed material to lysosomes.
Future improvements
I n c r e a s e d popularity of this technique will undoubtedly lead to
g r e a t e r e f f i c i e n c y and i m p r o v e d m e t h o d s .
If rapid delivery to
lysosomes is a problem for a certain cell type, then the uptake period
can be shortened providing that the protein's specific activity is high
enough to introduce sufficient radioactivity into the cells.
Recently, it was reported (81) that material pinocytosed in the
p r e s e n c e of adenovirus was released to the cytosol due to viralmediated rupture of endosomes.
It remains to be established if this
m e t h o d can circumvent some of the problems associated with the
present techniques.
Concluding Remarks
Because cell extracts can not always be coaxed into reproducing
complex cellular processes (3), microinjection can provide a method
for i n t r o d u c i n g s p e c i f i c molecules into functioning cells.
These
techniques can be used to directly address many questions concerning
protein actions, localization, and eventual catabolism within cells.
We look f o r w a r d to the application of microinjection to other
important questions in cell biology in the near future.
For example,
the s p e c i f i c proteins in m i t o t i c cells responsible for chromatin
condensation might be identified (82).
In addition, microinjection of
the putative 'trigger protein' which may regulate the cell cycle (83)
could determine whether increased levels are necessary for G0/G1-to-S
transition. Similar studies have already been undertaken to investigate
the role of a small nuclear RNA species in the inhibition of protein
synthesis that occurs during mitosis (80). Microinjection could also be
used to identify inhibitors of cell division that accumulate in the
cytoplasm of senescent cells (85). Whether the actions of polypeptide
hormones such as insulin are mediated through putative peptide
intermediates (86,87) can be addressed by microinjecting the intermediates.
Other
p o t e n t i a l applications of microinjection include
identification of rate-limiting steps in enzymatic pathways by increasing the amount of specific enzymes and measuring the effects on the
pathway; analysis of the assembly into mitochondria (88), peroxisome
( 8 9 ) , endoplasmic reticulum (89), and plasma membranes (90) of
proteins which do not require cotranslational insertion; and injection of
steroid-hormone receptors to investigate mechanisms of transfer into
the nucleus (91). It would be most interesting if responsiveness to a
steroid hormone can be transferred to a particular cell by injecting
the proper receptor.
M i c r o i n j e c t i o n m a y even have important medical applications.
E x p l o i t a t i o n of cell fusion and specific targeting techniques has
exciting implications for novel drug-delivery systems, enzyme replacem e n t for p a t i e n t s w i t h enzyme deficiencies, and s i t e - s p e c i f i c
MICROIN3ECTION
OF
CULTURED
CELLS
463
chemotherapy.
In the years to come, microinjection should provide
answers to many of the current questions about cell physiology as well
as opening new areas of inquiry.
Acknowledgements
The authors thank Dr. 3.3. Berger, E.A. Gulve, and S. Golf for
critically reading the manuscript. Research in the authors' laboratory
was supported by grants from the Muscular Dystrophy Association of
America, the American Heart Association, and the National Institutes
of Health (AG02783). MAM is the recipient of the Paul Dudley White
F e l l o w s h i p of the A m e r i c a n H e a r t Association and 3FD holds a
R e s e a r c h Career Development Award for the National Institute of
Aging.
References
i. Stacey DW & Allfrey Vg (1977) J. Cell Biol. 75, 807-817.
2. Graessmann A, Graessmann M & Mueller C (1979) Biochem.
Biophys. Res. Commun. 88, 428-432.
3. Kulka RG & Loyter A (1979) in: Current Topics in Membranes and
Transport, vol 12 (Bronner F & Kleinzeller A, eds), pp
366-430, Academic Press, Inc.
4. Furusawa M, Nishimura T, Yamaizumi M & Okada Y (1974) Nature
249, 449-450.
5. Schlegel RA & Rechsteiner MC (1975) Cell 5, 371-379.
6. Loyter A, Zakai N & Kulka RG (1975) J. Cell Biol. 66,
292-304.
7. Kimelberg HK & Mayhew E (1978) CRC Crit. Rev. Toxicol. 6,
25-79.
8. Pagano RE & Weinstein JN (1978) Annu. Rev. Biophys. Bioeng.
7, 435-468.
9. Okada CY & Rechsteiner M (1982) Cell 29, 33-41.
I0. Diacumakos EG (1980) in: Introduction of Macromolecules into
Viable Mammalian Cells, vol~l (Baserga R, Croce C & Rovera G,
eds), pp 85-98, Alan R Liss, Inc, New York.
ii. Diacumakos EG, Holland S & Pecora P (1970) Proc. Natl. Acad.
Sci. USA 65, 911-919.
12. Taber R, Tazewell W & Papahadjopoulos D (1980) in: Introduction of M~cromolecules into Viable Mammalian Cells, vol 1
(Baserga R, Croce C & Rovera G, eds), pp 221-238, Alan R Liss,
Inc, New York.
13. $zoka F, Jacobson K, Derzko Z & Papahajopoulos D (1980)
Biochim. Biophys. Acta 600, 1-18.
14. Neff NT, Bourret L, Miao P & Dice JF (1981) J. Cell Biol. 91,
184-194.
15. Dice JF (1982) J. Biol. Chem. 257, 14624-14627.
16. Backer JM, Bourret L & Dice JF (1983) Proc. Natl. Acad. Sci.
USA 80, 2166-2170,
17. McElligott MA & Dice JF (1983) Biochem. J. 216 559-566.
18. Ihler G, Glew RH & Schnure FW (1973) Proc. Natl. Acad. Sci.
70, 2663-2666.
19. Rechsteiner MC (1975) Exptl. Cell Res. 93, 487-492.
464
McELLIGOTT & DICE
20. Schlegel RA & Rechsteiner MC (1978) Meth~ Cell Biol. 20,
341-354.
21. Seeman P (1967) J. Cell Biol. 32, 55-70.
22. Kriegler MP & Livingston DM (1977) Somatic Cell Genetics 3,
603-610.
23. Kaltoft K, Zeuthen J, Engbaek F, Piper PW & Cells JE (1976)
Proc. Natl. Acad. Sci. USA 73, 2793-2797.
24. Asano K, Murachi T & Asano A (1983) J. Bioehem. 93, 733-741.
25. Kruse CA, Spector EB, Cederbaum SD, Wisnieski BJ & Popjak G
(1981) Biochim. Biophys. Acta 645, 339-345.
26. Vainstein A, Razin A, Graessman A & Loyter A (1983) Meth.
Enzymol. I01, 492-512.
27. Beigel M & Loyter A (1983) Exp. Cell Res. 148, 95-103.
28. Wojcieszyn JW, Schlegel RA, Lumley-Sapanski K & Jacobson KA
(1983) J. Cell Biol. 96, 151-159.
29. Boni LT, Stewart TP, Alderfer JL & Hui SW (1981) J. Memb.
Biol. 62, 65-70.
30. Maggio B, Ahkong QF & Lucy JA (1976) Biochem. J. 158, 647-650.
31. Knutton S (1979) J. Cell Sci. 36, 61-72.
32. Honda K, Maeda Y, Sasakawa S, Ohno H & Tsuchida E (1981)
Biochem. Biophys. Res. Commun. IO1, 165-171.
33. Smith CL, Ahkong QF, Fisher D & Lucy J (1982) Biochim.
Biophys. Acta 692, 109-114.
34. Bigelow S, Hough R & Rechsteiner M (1981) Cell 25, 83-93.
35. Dice JF, Bourret L & Miao P (1981) J. Cell Biol. 91, 241a.
36. McGarry T, Hough R, Rogers S & Rechsteiner M (1983) J. Cell
Biol. 96, 338-346.
37. Rechsteiner M, Chin D, Hough R, McGarry T, Rogers S, Rote K &
Wu L (1984) in: Cell Fusion, Ciba Foundation Symposium 103,
pp 181-195, Pitman, London.
38. Hendil KB (1980) J. Cell Physiol. IO5, 449-460.
39. Zavortink M, Thacher T & Rechsteiner M (1979) J. Cell Physiol.
100, 175-186.
40. Wasserman M, Kulka RG & Loyter A (1977) FEBS Lett. 83, 48-52.
41. Mayer RJ, Evans P, Russell S & Amenta JS (1984) in: Cell
Fusion, Ciba Foundation Symposium 103, pp 202-214, Pitman,
London.
42. Rechsteiner M & Kuehl L (1979) Cell 16, 901-908.
43. Atidia J & Kulka RG (1982) FEBS Lett. 142, 72-76.
44. Chin DT, Kuehl L & Rechsteiner M (1982) Proc. Natl. Acad. Sci.
79, 5857-5861.
45. Katznelson R & Kulka RG (1983) J. Biol. Chem. 258, 9597-9600.
46. Yamaizumi M, Uchida T, Mekada E & Okada Y (1979) Cell 18,
1009-1014.
47. Rote KV & Rechsteiner M (1983) J. Cell Physiol. 116, 103-111.
48. Hendil KB (1981) Exp. Cell Res. 135, 157-166.
49. Freikopf-Cassel A & Kulka RG (1981) FEBS Lett. 128, 63-66.
50. Wojcieszyn JW, Schlegel RA, Wu ES & Jacobson KA (1981) Proc.
Natl. Acad. Sci. USA 78, 4407-4410.
51. Bennett FC, Busch H, Lischwe MA & Yeoman LC (1983) J. Cell
Biol. 97, 1556-1572.
52. Wu L, Rechsteiner M & Kuehl L (1981) J. Cell Biol. 91,
488-496.
MICROIN3ECTION
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
OF
CULTURED
CELLS
t465
Boogaard C & Dixon GH (1983) Exp. Cell Res. 143, 191-205.
Boogaard C & Dixon GH (1983) Exp. Cell Res. 143, 175-190.
Kaltoft K & C e l i s JE (1978) Exp. Cell. Res. 115, 423"428.
Antman KH & Livingston DM (1980) Cell 19, 627-635.
Yamaizumi M, Uchida T, Okada Y & Furusawa M (1978) Cell 13,
227-232.
Boney C, Fink D, Schlichter D, Carr K & Wicks WD (1983) J.
Biol. Chem. 258, 4911-4918.
Ohara J, Sugi M, Fujimoto M & Watanabe T (1982) J. Immunol.
129, 1227-1232.
Ohara J & Watanabe T (1982) J. Immunol. 128, 1090-1096.
Kriegler MP, Griffen JD & Livingston DM (1978) Cell 14,
983-994.
Straus SE & Raskas HJ (1980) J. Gen. Virol. 48, 241-245.
Mercer WE, Terefinko DI & Schlegel RA (1979) Cell Biol. Intl.
Repts. 3, 265-270.
Kriegler MP & Livingston DM (1980) in: Introduction of
Macromolecules into Viable Mammalian Cells, vol 1 (Baserga R,
Croce C & Rovera G, eds) pp 261-296, Alan R Liss, Inc, New York.
Kishimoto T, Miki Y, Kishi H, Muraguchi A, Kishimoto S &
Yamamura Y (1982) J. Immunol. 129, 1367-1371.
Morrison M & Sehonbaum GR (1976) Ann. Rev. Biochem. 45,
861-888.
Kim S & P a i k W K (1965) J. Biol. Chem. 240, 4629-4634.
Tweto J & Doyle D (1976) J. Biol. Chem. 251, 872-882.
Evans PJ & Mayer RJ (1982) Biochem. Biophys. Res. Commun.
107, 51-58.
Dice JF, Hess EJ & Goldberg AL (1979) Biochem. J. 178,
305-312.
Furusawa M (1980) Int. Rev. Cytol. 62, 29-67.
Docherty PA & Aronson NN (1982) Biochem. Biophys. Res. Commun.
109, 527-532.
Wasserman M, Zakai N, Loyter A & Kulka RG (1976) Cell 7,
551-556.
Smith JD & Liu AY-C (1981) Sei. 214, 799-800.
Godfrey W, Doe B & Wofsy L (1983) Proc. Natl. Acad. Sci. USA
80, 2267-2271.
Brown DB, Hanks SK & Rao PN (1981) J. Cell Biol. 91, 12a.
Zimmerman U & Vienken J (1982) J. Memb. Biol. 67, 165-182.
Steinman RM, Mellman IS, Muller WA & Cohn ZA (1983) J. Cell
Biol. 96, 1-27.
Merion M & Sly WS (1983) J. Cell Biol. 96, 644-650.
Storrie B, Pool RR Jr, Sachdera M, Maurey KM & Oliver C (1984)
J. Cell. Biol. 98, 108-115.
FitzGerald DJP, Padmanabhan R, Pastan I & Willingham MC (1983)
Cell 32, 607-617.
Johnson RT & Rao PN (1970) Nature 226, 717-722.
Croy RG & Pardee AB (1983) Proc. Natl. Acad. Sci. 80,
4699-4703.
Smith JH, Subbarao MN & Eliceiri GL (1983) J. Cell. Phys.
114, 1-6.
Drescher-Lincoln CK & Smith JR (1983) Expt. Cell Res. 144,
455-462.
466
McELLIGOTT
&
DICE
86. Larner J, Cheng K, Schwartz C, Kikuchi K, Tamura S, Creacy S,
Dubler R, Galasko G, Pullin C & Katz M (1982) Fed. Proc. 41,
2724-2729.
87. Seals JR & Czech MP (1982) Fed. Proc. 41, 2730-2735.
88. Schatz G & Butow RA (1983) Cell 32, 316-318.
89. Sabitini DD, Kreibich G, Morimoto T & Adesnik M (1982) J. Cell
Biol. 92, 1-22.
90. Levinson AD, Courtneidge SA & Bishop JM (1981) Proc. Natl.
Acad. Sci. 78, 1624-1628.
91. Tepperman J (1980) in: Metabolic and Endocrine Physiology,
Fourth Edition, pp 21-28, Year Book Medical Publishers, Inc.,
Chicago, London.