1 Structural Biology Laboratory, The Salk Institute for Biological Studies, La
Jolla, CA 92037, USA
2 Department of Molecular, Cellular and Developmental Biology, Yale University,
New Haven, CT 06520-8103, USA
* Present address: Department of Parasitology, Kyungpook National University
School of Medicine, Taegu 700-422, Korea
Author for correspondence (e-mail:
thomas.pollard{at}yale.edu)
Accepted 4 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Myosin, Motility, Microscopy, Green fluorescent protein, Transfection
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myosin dynamics have been documented in animal cells
(Wei and Adelstein, 2000;
Buss et al., 2001
;
Tang and Ostap, 2001
),
Dictyostelium (Moores et al.,
1996
; Zang and Spudich,
1998
; Yumura,
2001
; Neujahr et al.,
1997
) and yeast (Bezanilla et
al., 2000
; Lee et al.,
2000
) by expressing myosin fusion proteins tagged with green
fluorescent protein (GFP). Such GFP fusion proteins can take the place of
wild-type Dictyostelium myosin-II
(Moores et al., 1996
), yeast
myosin-II (Bezanilla et al.,
2000
) and yeast myosin-I (Lee
et al., 2000
). Analysis of the cellular dynamics of the
biochemically well-characterized Acanthamoeba myosins has been
hampered by the lack of a robust transfection system. Hu and Henney
(Hu and Henney, 1997
) had some
success transfecting Acanthamoeba by electroporation with a CAT
(chloramphenicol acetyltransferase) reporter gene under the control of the
amoeba ubiquitin promoter. We have improved the transfection method for
Acanthamoeba, so that transient expression of a variety of GFP-fusion
proteins is now routine.
We find that myosin-II assembles into particles of two sizes, interpreted as mini-filaments and thick filaments. Thick filaments form anteriorly and laterally and then move to the rear of the cells where they disassemble. In cells also expressing full length myosin-II, only the C-terminal 256 residues of the myosin-II tail are required for the GFP-fusion protein to go through this cycle of assembly and disassembly. Mutation of the inhibitory phosphorylation sites in the tail piece of myosin-II to alanine resulted in abnormal accumulation of the construct around vacuoles in the cell. Myosin-IC accumulates around contractile vacuoles seconds before they discharge, as well as around macropinocytosis cups just before they pinch off. Localization to these two sites requires both the head of the myosin and most of the tail, including the SH3 domain.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Transfection
Acanthamoeba castellanii Neff strain grown to mid log phase in a
bubbler at room temperature were washed twice with PBS (8 g NaCl, 0.2 g KCl,
1.44 g Na2HPO4, 0.24 g KH2PO4 per
liter of water, pH 7.4) and resuspended in PYG culture medium (20 g Proteose
Peptone, 1 g yeast extract, 4 mM MgSO4, 0.4 mM CaCl2,
3.4 mM sodium citrate, 0.05 mM
Fe(NH4)2(SO4)2, 2.5 mM
Na2HPO4, 2.5 M KH2PO4, pH 6.5, in
950 ml deionized water and autoclaved followed by addition of 50 ml of
sterile-filtered 2 M glucose). Cells were cultured overnight at 25°C in a
6-well culture plate with 4x105 cells per well in 3 ml of
culture medium. Four micrograms of plasmid DNA in 100 µl of amoeba culture
medium were mixed with 20 µl of Superfect (Qiagen), incubated for 10
minutes at room temperature and then diluted with 600 µl of culture medium.
Adherent amoeba in a 6-well plastic culture plate were washed once with room
temperature PBS. After removing most of PBS, DNA-Superfect mixture was added
drop-wise to the cells. Cells were incubated at 25°C for 3 hours to allow
uptake of DNA-Superfect complexes. Cells were washed once with PBS,
resuspended in 3 ml of fresh growth medium and incubated at 25°C for 24-48
hours. Expression of EGFP was checked by fluorescence microscopy.
Flow cytometry and cell sorting
Cells were washed twice and resuspended in 2 ml of PBS. EGFP expression of
1x106 cells was measured with a CellQuest 3.2 FACScan
(Becton-Dickinson). For microscopic examination and immunoblots, transfected
cells expressing EGFP were isolated by fluorescence-activated cell sorting
using a Facstar (Becton-Dickinson) with a single laser.
Microscopy
Sorted amoeba expressing green fluorescent protein were allowed to adhere
to a glass-bottomed microwell during incubation for 1-2 hours at room
temperature in PBS. Alternatively, transfected amoebae were flattened with an
agar overlay (Yumura et al.,
1984). Excess PBS was absorbed from the edge of the agar slice
with filter paper until the cells flattened appropriately. To increase the
frequency of contractile vacuole discharge, cells were washed briefly in PBS,
1:1 PBS:water, and incubated in water for 10 minutes at room temperature. Live
cells were imaged with an Olympus IX70 fluorescent microscope with 100x
objective, cooled CCD camera and motorized Z-axis stage. Images were processed
with Photoshop 5.5.
Immunoblots
One thousand amoeba sorted by their green fluorescence were boiled in
SDS-PAGE sample buffer, electrophoresed on a 15% acrylamide gel and blotted on
a nylon membrane. Blotted proteins were detected with the following
antibodies: a mixture of mouse monoclonal antibodies that react with
Acanthamoeba profilin-I and profilin-II
(Kaiser and Pollard, 1996); a
mixture of 13 monoclonal antibodies specific for the C-terminal region of the
Acanthamoeba myosin-II tail
(Kiehart et al., 1984
); actin
monoclonal antibody c4d6 (Lessard,
1988
); and mouse monoclonal anti-GFP (Zymed, San Francisco, CA).
To reduce non-specific reactions, the anti-GFP antibody was preabsorbed with
untransfected cells prepared by methanol fixation. Bound antibodies were
detected by chemiluminescence.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We used profilin-I-EGFP to optimize the transfection conditions. Superfect
was much more efficient than electroporation or any other method that we
tested. Fresh amoeba and fresh DNA gave the best results. More cells were
transfected with 4 µg of plasmid DNA in Superfect than Hu and Henney
(Hu and Henney, 1997) reported
with electroporation of 100 µg of DNA. In our hands electroporation
produced very few transfected cells. The codon usage of EGFP is closer to that
of Acanthamoeba than GFP, but modifying three EGFP codons that are
rarely, if ever, used by Acanthamoeba might improve further the
performance of the vector.
When expression was quantitated by flow cytometry, the EGFP fluorescence of individual cells varied in intensity by three orders of magnitude for any given expression vector (Fig. 2). Cells in the large peak with fluorescence <50 did not express EGFP. Cells in fraction M2 (fluorescence 90-1000) were alive and suitable for microscopy. The M2 fraction of cells expressing EGFP-profilin grew at the same rate as control cells, but over two weeks all of the cells lost their green fluorescence, probably owing to loss of the plasmid. The M3 fraction of cells with fluorescence >1000 were round and immotile. The proportion of M3 cells was much larger in populations expressing EGFP-profilin than other constructs. These highly fluorescent cells rounded up before cell sorting, so their inactivity did not result from sorting.
Quantitation of expression levels showed that the highest levels achieved were in the micromolar range, below the concentrations of the endogenous native proteins, so the EGFP-fusion proteins were tracers in a population of these native proteins. We used cells expressing EGFP-profilin and EGFP-myosin-II tail fragment for quantitating expression, since they were expressed at higher levels than the larger constructs. We isolated transfected cells by cell sorting and compared the levels of expression of the fusion protein with the endogenous protein. Quantitative immunoblots (Fig. 3) showed that the cellular concentration of profilin-I-EGFP in the combined M2 and M3 fractions was 10% that of endogenous profilins (or 13% of the profilin-I), or about 10 µM (that is 10 nmoles/g packed cells). Given the relative numbers of cells in M2 and M3 and their fluorescence (Fig. 2), M2 cells expressed 0.3 to 3 µM EGFP-profilin-I, while M3 cells expressed 3.5 to 35 µM EGFP-profilin-I. The average cellular concentration of EGFP-myosin-II tail fragment in the combined M2 and M3 fractions was about 20% that of endogenous myosin-II, or 0.2 µM. These EGFP-myosin-II tail fragment samples had few cells in M3, so the cells used for microscopy expressed on average about 0.1 µM fusion protein. In accordance with these measurements, cells expressing EGFP-profilin were moderately brighter by fluorescence microscopy than cells expressing EGFP-myosin-II tail. By both flow cytometry and fluorescence microscopy the expression of EGFP-myosin-II tail fragment was higher than the larger myosin fusion proteins. Thus, smaller constructs not only transfected a higher fraction of the cells but also were expressed at higher levels in these cells.
|
We placed EGFP at the N-termini of our myosin constructs, since N-terminal
GFP-myosin-II complements null mutations in Dictyostelium
(Moores et al., 1996;
Zang and Spudich, 1998
) and
S. pombe (Bezanilla et al.,
2000
) and N-terminal GFP-myosin-I complements null mutations in
S. pombe (Lee et al.,
2000
).
Distribution of EGFP and profilin-I-EGFP in live cells
Profilin-I-EGFP served as a control for our expressed myosin fusion
proteins in live amoebas, since we could compare its behavior with that of
rhodamine-labeled profilin-II introduced by syringe loading
(Kaiser et al., 1999). The
fluorescence of profilin-I-EGFP in live amoebas
(Fig. 4A,B) was very similar to
that of rhodamine-profilin-II and distinctly different from free EGFP.
Profilin-I-EGFP filled the cytoplasm but was excluded from organelles
including the nucleus. In contrast, free EGFP accumulated in the nucleus
except for the nucleolus (Fig.
4C,D). Profilin-I-EGFP concentrated in pseudopods to a greater
extent than free EGFP.
|
Distribution of EGFP-myosin-II in live cells
We studied myosin-II dynamics in live cells using two different EGFP-fusion
proteins. The fluorescence was similar in cells expressing EGFP-full length
myosin-II (Fig. 5A) and EGFP
fused to the C-terminal 256 residues of the myosin-II tail
(Fig. 5B,C). The EGFP-myosin-II
tail construct produced better images, owing to the higher transfection
efficiency and expression level (about 10% of the endogenous native myosin-II)
compared with longer constructs. Our use of trace quantities of EGFP-myosin-II
tail construct as a probe for myosin-II dynamics in vivo is justified by
detailed studies of the assembly properties of the myosin-II tail. Truncations
of the distal part of the myosin-II tail
(Sinard et al., 1990) and
experiments with monoclonal antibodies with carefully mapped epitopes
(Rimm et al., 1990
) showed
that the C-terminal 100 residues are required for assembly. Biophysical
analysis of purified myosin-II constructs has established that the C-terminal
256 residues of the myosin-II tail are sufficient to assemble an octamer
equivalent to minifilaments of the full length protein (K. Turbedsky, Assembly
of Acanthamoeba myosin-II, PhD thesis, Johns Hopkins University,
2001; K. Turbedsky and T. D. Pollard, unpublished).
|
EGFP-full length myosin-II and EGFP-myosin-II tail were confined to the
cytoplasm and concentrated in spots of two sizes. The large spots were
asymmetric with dimensions of up to 0.4x0.8 µm, the same size as
fluorescence images of thick filaments of purified myosin-II and myosin-II in
fixed cells stained with a fluorescent monoclonal antibody
(Yonemura and Pollard, 1992).
Thick filaments are actually much smaller than their fluorescence images,
being bipolar rods about 20 nm in diameter and 300 nm long
(Sinard and Pollard, 1989
).
Smaller spots, presumed to be myosin-II minifilaments, were scattered
throughout the cytoplasm but rarely at the very front of the cell. Small spots
are readily visible as bright flecks when focusing through a cell with the
microscope but difficult to reproduce in printed micrographs. Flattening
transfected cells with an agar coverslip reduced superimposition
(Yumura et al., 1984
) and
improved the resolution of both types of spots. Compression distorts the 3D
architecture of the cell and is thought to stress Dictyostelium
(Neujahr et al., 1997
), but
the behavior of EGFP-myosin-II was similar in most respects in 3D and
flattened Acanthamoeba.
Large spots of EGFP-myosin-II concentrated at the trailing edge of cells (Figs 5,6,7) and spread out over the dorsal surface of unflattened cells (Fig. 6). Few large spots were present near the bottom surface. Small fluorescent spots of EGFP-myosin-II were more abundant in the internal cytoplasm than the cortex. In some transfected amoeba, large myosin-II spots aggregated at random positions including the posterior, anterior or mid-regions of the cytoplasm (Fig. 5B, Fig. 7A). These aggregates moved freely through the cytoplasm. Compression and/or the expression of the tail construct may have contributed to these artifactual accumulations of myosin-II.
|
|
Time-lapse records (Fig. 5B,C and see movie online; Fig. 7) revealed two types of myosin-II dynamics. The large spots of myosin-II underwent a cycle of assembly and disassembly on a time scale of tens of seconds. Large spots of myosin-II appeared de novo and grew into large fluorescent spots over about 60 seconds. In moving cells, most of the large spots appeared near the front or along the sides. Thick filaments disappeared by losing their fluorescent intensity over about 60 seconds, most commonly near the rear of the cell (Fig. 7B). This represented disassembly rather than either (1) movement to another plane of focus (since the entire thickness of these flattened cells was in focus) or (2) photobleaching (since the fluorescence of other nearby filaments was stable).
Both types of EGFP-myosin-II spots moved continuously through the cytoplasm. Thick filaments moved from their sites of formation toward the rear of the cell. Some large spots increased in intensity as they moved backward. Those in Fig. 5B,C [see movie online (http://jcs.biologists.org/supplemental)] moved at about 25 nm/second, but their velocities varied within each cell. These movements resulted in the accumulation of bright spots at the rear where disassembly also took place. Small fluorescent spots in the deep cytoplasm moved forward. When an amoeba changed direction, the flow of spots reversed and thick filaments began to accumulate at the new trailing edge within seconds (Fig. 7A).
Phosphorylation of three serines in the tail piece of Acanthamoeba
myosin-II inhibits the actin-activated ATPase in the heads
(Kuznicki et al., 1983). We
prepared a mutant EGFP-myosin-II, designed to be constitutively active through
replacement of all three inhibitory heavy chain phosphorylation sites with
alanine. Expression of this mutant myosin-II in cells containing wild-type
myosin-II produced large and small fluorescent spots, similar to the spots of
full length EGFP-myosin-II with wild-type phosphorylation sites, but the
distribution of the spots was altered (Fig.
8). The large spots of constitutively active EGFP-myosin-II
accumulated like a sunburst around vesicles, likely to be endocytic in origin.
Some of these decorated vesicles fused. Wild-type EGFP-myosin-II never
associated with such vesicles.
|
Distribution and dynamics of myosin-IC in live cells
EGFP-full length myosin-IC was distributed throughout the cytoplasm but
concentrated in lamellipodia at the leading edge of moving cells and around
contractile vacuoles and macropinocytic cups
(Fig. 9). EGFP-myosin-IC
accumulated transiently around contractile vacuoles only when they reached
their maximal size, about 15 to 30 seconds before they discharged their
contents, whereupon myosin-IC dispersed immediately
(Fig. 10). Baines and Korn
(Baines and Korn, 1995) reported that antibodies to myosin-IC stained the
contractile vacuole in fixed cells throughout its cycle and that this
myosin-IC was much more heavily phosphorylated at the time of contraction. The
contrast in fixed cells may have been greater owing to some extraction of
myosin-IC from the cytoplasm during fixation or permeabilization. The brief
concentration around the contractile vacuole at the time of contraction might
have been missed in fixed cells. We also found that EGFP-myosin-IC also
accumulated around macropinocytic cups in live cells 3 to 5 seconds before
their closure and then dispersed (Figs
9,
10). Myosin-IC was excluded
from organelles and the nucleus.
|
|
Expression of EGFP fused to N- or C-terminal truncation constructs showed that both the myosin head and the SH3 domain in the tail are required for myosin-IC to localize to contractile vacuoles and macropinocytic cups (Fig. 11). Over 30 cells were observed for each construct. The EGFP construct lacking just the C-terminal GPA domain (MICs) localized much like the full length construct in the cytoplasm and around contractile vacuoles and macropinocytic cups. EGFP-myosin-IC constructs lacking the SH3 (MICg) or GPA and SH3 (MICb) domains of the tail were distributed throughout the cytoplasm and did not concentrate around contractile vacuoles or macropinocytic cups. Constructs consisting of head (MICh) or tail alone (not shown) did not localize around contractile vacuoles or macropinocytic cups. However, all truncated myosin-IC constructs, even those lacking the SH3 domain or head, did concentrate in lamellipodia. Contractile vacuoles filled and contracted normally in cells expressing each of the myosin-IC constructs.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of EGFP-myosin-II allowed us to observe a cycle of assembly and
disassembly of thick filaments in Acanthamoeba for the first time.
Thick filaments concentrated in the dorsal cortex and along the lateral edge
in the back half of the cells, while minifilaments were dispersed more
uniformly throughout the cytoplasm. Thick filaments form and grow in size over
a period of seconds. New thick filaments usually assemble behind the leading
edge and then move dorsally and laterally by cortical flow toward the rear of
the cell where they disassemble. Given the stability of mini-filaments under
physiological conditions (Sinard and
Pollard, 1989), we suggest that thick filaments disassemble into
minifilaments, which then move forward by bulk cytoplasmic flow for reassembly
in more anterior regions of the cell. However, mini-filaments are too small to
follow individually with enough confidence to be certain about their
stability. Millimolar concentrations of divalent cations and acid pH favor the
lateral association of pure myosin-II mini-filaments into thick filaments
(Sinard et al., 1990
). In
vivo, it seems more likely that yet to be identified accessory proteins rather
than ionic conditions regulate thick filament assembly.
Mutation of three phosphorylatable serines in the non-helical tail piece
had a greater impact on the localization of myosin-II than removing the head
and proximal tail. EGFP fused to the C-terminal 256 residues of myosin-II
behaved just like full length myosin-II, most likely owing to co-assembly with
the more abundant wild-type myosin-II. This tail construct include all of the
elements required to assemble octameric minifilaments in vitro (K. Turbedsky,
PhD thesis, Johns Hopkins University, 2001) and may suffice to specify many
functions. By contrast, expression of a low mole fraction of
unphosphorylatable myosin-II caused the filaments to cluster around vesicles
more than wild-type myosin-II. Three serines in the tail piece are heavily
phosphorylated in vivo (Collins and Korn,
1981). Although phosphorylation of the tail piece serines has no
effect on assembly of mini-filaments or thick filaments
(Sinard and Pollard, 1989
;
Redowicz et al., 1994
), it
does inhibit binding to actin filaments and the actin-activated ATPase of the
heads (Cote et al., 1981
;
Collins et al., 1982a
;
Collins et al., 1982b
;
Ganguly et al., 1992
).
Dephosphorylation allows actin filaments to activate the ATPase activity, and
so the triple alanine mutant is expected to be constitutively active.
Constitutively active myosin may bind to actin filaments associated with
cytoplasmic vesicles.
Comparison with the behavior of myosin-II in other cells
Although the best characterized cytoplasmic myosin-II filaments (from
vertebrate cultured cells, Dictyostelium and Acanthamoeba)
appear to differ in the mechanisms regulating their assembly and localization,
a few general themes are now apparent. Differences include regulation of
assembly by heavy chain phosphorylation in Dictyostelium
(Kolman et al., 1996) but not
the other systems, regulation of assembly by phosphorylation of the regulatory
light chain in vertebrate cells (Kolega
and Kumar, 1999
) but not the other systems, and the association of
myosin-II filaments with stress fibers in slowly moving vertebrate cells
(Kolega and Taylor, 1993
) but
not in rapidly moving cells. Nevertheless, in all three systems myosin-II
filaments concentrate at the rear of motile cells
(Moores et al., 1996
;
Clow and McNally, 1999
), where
they generate retraction forces. The following paragraphs discuss two other
features that these systems have in common.
Lateral clustering of minifilaments
Acanthamoeba and vertebrate myosin-II form stable mini-filaments
that associate laterally into larger assemblies both in vitro
(Sinard et al., 1989;
Niederman and Pollard, 1975
)
and in cells (Yonemura and Pollard,
1992
; Svitkina et al.,
1997
; current report). The function of these larger assemblies has
not been established experimentally, but is probably related to the greater
force produced by bringing together many heads in one physical unit. The
assembly mechanism of Dictyostelium myosin-II is less well
understood, but the appearance of large fluorescent spots in cells stimulated
with cAMP (Yumura et al.,
1984
) may also represent assembly of thick filaments from
mini-filaments or smaller oligomers.
Rapid recycling of myosin filaments
Migrating human fibroblasts (Kolega and
Taylor, 1993; DeBiasio et al.,
1996
) and fish epidermal keratocytes
(Svitkina et al., 1997
)
assemble myosin-II filaments in deep parts of lamellipodia. In keratocytes
discrete clusters of bipolar minifilaments increase in size and density
towards the peri-nuclear area and concentrate in transverse actin filament
bundles. These myosin clusters remain stationary with respect to the
substratum in locomoting cells, but they exhibit retrograde flow in cells
tethered in epithelioid colonies. The myosin-II assembly cycle is similar in
Acanthamoeba. Thick filaments form anteriorly and move toward the
rear where they disassemble. Thick filaments appear to assemble from and
disassemble into mini-filaments, but the regulatory mechanisms have yet to be
identified. Dictyostelium myosin-II filaments also turn over rapidly
with a half-life of 7 seconds in the cleavage furrow
(Yumura, 2001
). Reversible
phosphorylation of the Dictyostelium myosin-II heavy chain regulates
assembly in vitro (Kuczmarski and Spudich,
1980
) and in vivo (Kolman et
al., 1996
; Yumura,
2001
). PAK (p21 activated kinase) also influences the assembly and
localization of Dictyostelium myosin-II
(Chang and Firtel, 1999
).
Structural requirements for transient association of myosin-IC with
contractile vacuoles and macropinocytic cups
The bulk of EGFP-myosin-IC spreads uniformly throughout the cytoplasm, but
it concentrates for a few seconds around contractile vacuoles prior to their
discharge and on the cytoplasmic surface of the plasma membrane participating
in macropinocytosis prior to closure. In both cases myosin-IC dissipates
quickly after membrane fusion. More EGPF-myosin-IC appeared in the cytoplasm
between the organelles of live cells than in fixed cells
(Baines and Korn, 1990;
Baines et al., 1995
). Although
the role of myosin-IC in contractile vacuole function has yet to be defined,
it is the only myosin-I isoform detected around contractile vacuoles with
antibody staining (Baines and Korn,
1990
; Baines et al.,
1995
). Microinjection of antibodies to myosin-IC inhibited
contractile vacuole function, while injection of antibodies to myosin-IA or
-IB did not (Doberstein et al.,
1993
). In parallel work we labeled a monoclonal antibody specific
for myosin-IA and -IC with the fluorescent dye Cy3 and introduced it into live
cells by syringe loading (Ostap et al.,
2003
). This non-inhibitory fluorescent antibody associated
transiently with macropinocytosis cups and to a lesser extent with contractile
vacuoles. Comparison with EGFP-myosin-IC in this study suggests that myosin-IA
is more enriched around macropinocytosis cups and myosin-IC is enriched around
contractile vacuoles.
Five different truncation mutants revealed that localization of myosin-IC
to contractile vacuoles and macropinocytosis cups requires both the heads and
the SH3 domain in the tail, while the second TH2 domain at the C-terminus is
dispensable. In Acanthamoeba myosin-I heads provide ATP-sensitive
actin binding, the TH1 domains interact with both acidic lipids
(Doberstein and Pollard, 1992)
and actin filaments (Lee et al.,
1999
), TH2 domains reinforce actin filament binding
(Lynch et al., 1986
;
Lee et al., 1999
) and SH3
domains associate with Acan125 (Xu et al.,
1995
; Xu et al.,
1997
; Lee et al.,
1999
), the founding member of the CARMIL family of adapter
proteins. The Dictyostelium CARMIL protein interacts with both Arp2/3
complex and heterodimeric capping protein
(Jung et al., 2001
). Since the
SH3 domains of both myosin-IA and -IC bind Acan125 with high affinity
(Lee et al., 1999
;
Zot et al., 2000
), other
determinants must contribute to the distinct localizations of these two
myosin-I isoforms (Baines et al.,
1995
).
The available evidence suggests that the head and tail domains required for
biological function vary among myosin-Is from various species. Vertebrate
myo1b requires both heads and tail domains for localization to membrane
ruffles (Tang and Ostap,
2001). In Dictyostelium, expression of a mutant myoB
lacking the SH3 domain failed to rescue the defects in
myoA-/myoB- double mutants
(Novak and Titus, 1998
),
although Dictyostelium myoB lacking the SH3 domain localized
normally. In Aspergillus, deletion of the SH3 domain of MYOA has no
effect on cell growth, morphology, secretion or endocytosis
(Osherov et al., 1998
), while
the SH3 domain of budding yeast Myo5p is required for full function and
subcellular targeting (Anderson et al.,
1998
). The SH3 domains of budding yeast myosin-Is participate in
actin assembly by binding the actin-associated protein verprolin, the fungal
homolog of WASp-interacting protein, WIP
(Roberson et al., 1997
;
Lechler et al., 2000
;
Evangelista et al., 2000
). By
contrast, only the head and basic region (TH1) of the single fission yeast
myosin-I, Myo1p, are required for cortical localization and to complement the
defects of myo1 deletion (Lee et al.,
2000
). However, the SH3 domain of Myo1p is required for full
function in the absence of the WASp homolog Wsp1p. Since a consistent picture
of the relation of tail domains to function has yet to emerge, these myosin-Is
may genuinely differ from each other.
Insights from the fungal systems and the discovery of the CARMIL family of adapter proteins emphasize that myosin-I is part of a complex network of interacting proteins that regulate actin assembly. Further work is required to learn how the motile activities and actin assembly activities of the various myosin-I filaments contribute to their biological functions. Such efforts to relate structure and function will have to take into account all of the components of these systems.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, B. L., Boldogh, I., Evangelista, M., Boone, C.,
Greene, L. A. and Pon, L. A. (1998). The src homology domain
3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required
for targeting to sites of actin polarization. J. Cell
Biol. 141,1357
-1370.
Baines, I. C. and Korn, E. D. (1990). Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy. J. Cell Biol. 111,895 -904.
Baines, I. C., Corigliano-Murphy, A. and Korn, E. D. (1995). Quantification and localization of phosphorylated myosin-I isoforms in Acanthamoeba. J. Cell Biol. 130,591 -603.[Abstract]
Bezanilla, M., Wilson, J. M. and Pollard, T. D. (2000). Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis. Curr. Biol. 6,397 -400.
Brzeska, H., Kulesza-Lipka, D. and Korn, E. D.
(1992). Inhibition of Acanthamoeba myosin I heavy chain
kinase by Ca2+-calmodulin. J. Biol. Chem.
267,23870
-23875.
Buss, F., Arden, S. D., Lindsay, M., Luzio, J. P. and
Kendrick-Jones, J. (2001). Myosin VI isoform localized to
clathrin-coated vesicles with a role in clathrin-mediated endocytosis.
EMBO J. 20,3676
-3684.
Chang, Y. C. and Firtel, R. A. (1999). PAKa, a
putative PAK family member, is required for cytokinesis and the regulation of
the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.
J. Cell Biol. 147,559
-575.
Clow, P. A. and McNally, J. G. (1999). In vivo
observations of myosin II dynamics support a role in rear retraction.
Mol. Biol. Cell 10,1309
-1323.
Collins, J. H. and Korn, E. D. (1981).
Purification and characterization of actin-activatable, Ca2+ sensitive myosin
II from Acanthamoeba. J. Biol. Chem.
256,2586
-2595.
Collins, J. H., Cote, G. P. and Korn, E. D.
(1982a). Localization of the three phosphorylation sites on each
heavy chain of Acanthamoeba myosin-II to a segment at the end of the
tail. J. Biol. Chem.
257,4529
-4534.
Collins, J. H., Kuznicki, J., Bowers, B. and Korn, E. D. (1982b). Comparison of the actin binding and filament formation properties of phosphorylated and dephosphorylated Acanthamoeba myosin-II. Biochemistry 21,6910 -6915.[Medline]
Cote, G. P., Collins, J. H. and Korn, E. D.
(1981). Identification of three phosphorylation sites on each
heavy chain of Acanthamoeba myosin II. J. Biol.
Chem. 256,12811
-12816.
DeBiasio, R. L., LaRocca, G. M., Post, P. L. and Taylor, D. L. (1996). Myosin II transport, organization, and phosphorylation: Evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion. Mol. Biol. Cell 7,1259 -1282.[Abstract]
Doberstein, S. K., Baines, I. C., Wiegand, G., Korn, E. D. and Pollard, T. D. (1993). Inhibition of contractile vacuole function in vivo by antibodies against myosin-I. Nature 365,841 -843.[CrossRef][Medline]
Doberstein, S. K. and Pollard, T. D. (1992). Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC. J. Cell Biol. 117,1241 -1249.[Abstract]
Evangelista, M., Klebl, B. M., Tong, A. H. Y., Webb, B. A.,
Leeuw, T., Leberer, E., Whiteway, M., Thomas, D. Y. and Boone, C.
(2000). A role for myosin-I in actin assembly through
interactions with Vrp1p, Bee1p, and the Arp2/3 complex. J. Cell
Biol. 148,353
-362.
Ganguly, C., Baines, I. C., Korn, E. D. and Sellers, J.
(1992). Regulation of the actin-activated ATPase and in vitro
motility activities of monomeric and filamentous Acanthamoeba myosin
II. J. Biol. Chem. 267,20900
-20904.
Hu, Q. and Henney, H. R., Jr (1997). An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system. Biochim. Biophys. Acta 1351,126 -136.[Medline]
Jung, G., Remmert, K., Wu, X., Volosky, J. M. and Hammer, J. A.,
III (2001). The Dictyostelium CARMIL protein links
capping protein and the Arp2/3 complex to type I myosins through their SH3
domains. J. Cell Biol.
153,1479
-1497.
Kaiser, D. A. and Pollard, T. D. (1996). Characterization of actin and poly-L-proline binding sites of Acanthamoeba profilin with monoclonal antibodies and by mutagenesis. J. Mol. Biol. 256,89 -107.[CrossRef][Medline]
Kaiser, D. A., Vinson, V. K., Murphy, D. B. and Pollard, T.
D. (1999). Profilin is predominantly associated with
monomeric actin in Acanthamoeba. J. Cell Sci.
112,3779
-3790.
Kiehart, D. P., Kaiser, D. and Pollard, T. D. (1984). Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail. J. Cell Biol. 99,1015 -1023.[Abstract]
Kolega, J. and Kumar, S. (1999). Regulatory light chain phosphorylation and the assembly of myosin II into the cytoskeleton of microcapillary endothelial cells. Cell Motil. Cytoskel. 43,255 -268.[CrossRef][Medline]
Kolega, J. and Taylor, D. L. (1993). Gradients in the concentration and assembly of myosin II in living fibroblasts during locomotion and fiber transport. Mol. Biol. Cell 4, 819-836.[Abstract]
Kolman, M. F., Futey, L. M. and Egelhoff, T. T. (1996). Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development. J. Cell Biol. 132,101 -109.[Abstract]
Kuczmarski, E. R. and Spudich, J. A. (1980). Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. Proc. Nat. Acad. Sci. USA 77,7292 -7296.[Abstract]
Kuznicki, J., Albanesi, J. P., Cote, J. P. and Korn, E. D.
(1983). Supramolecular regulation of the actin-activated ATPase
activity of filaments of Acanthamoeba myosin-II. J. Biol.
Chem. 258,6011
-6014.
Lechler, T., Shevchenko, A., Shevchenko, A. and Li, R.
(2000). Direct involvement of yeast type 1 myosins in
Cdc42-dependent actin polymerization. J. Cell Biol.
148,363
-373.
Lee, W., Ostap, E. M., Zot, H. G. and Pollard, T. D.
(1999). Organization and ligand binding properties of the tail of
Acanthamoeba myosin-IA. J. Biol. Chem.
274,35159
-35171.
Lee, W. L., Bezanilla, M. and Pollard, T. D.
(2000). Fission yeast myosin-I, Myo1p, stimulates actin assembly
by Arp2/3 complex and shares functions with WASp. J. Cell
Biol. 151,789
-799.
Lessard, J. (1988). Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil. Cytoskeleton 10,349 -362.[Medline]
Lynch, T. J., Albanesi, J. P., Korn, E. D., Robinson, E. A.,
Bowers, B. and Fujisaki, H. (1986). ATPase activities and
actin-binding properties of subfragments of Acanthamoeba myosin-IA.
J. Biol. Chem. 261,17156
-17162.
Moores, S. L., Sabry, J. H. and Spudich, J. A.
(1996). Myosin dynamics in live cells. Proc. Natl.
Acad. Sci. USA 93,443
-446.
Neujahr, R., Heizer, C., Albrecht, R., Ecke, M., Schwartz, J.
M., Weber, I. and Gerisch, G. (1997). Three-dimensional
patterns and redistributions of myosin II and actin in mitotic
Dictyostelium cells. J. Cell Biol.
139,1793
-1804.
Niederman, R. and Pollard, T. D. (1975). Human platelet myosin. II. In vitro assembly of myosin and structure of myosin filaments. J. Cell Biol. 67, 72-92.[Abstract]
Novak, K. D. and Titus, M. A. (1998). The
myosin I SH3 domain and TEDS rule phosphorylation site are required for in
vivo function. Mol. Biol. Cell
9, 75-88.
Osherov, N., Yamashita, R. A., Chung, Y. S. and May, G. S.
(1998). Structural requirements for in vivo myosin I function in
Aspergillus nidulans. J. Biol. Chem.
273,27017
-27025.
Ostap, E. M. and Pollard, T. D. (1996). Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase. J. Cell Biol. 132,1053 -1060.[Abstract]
Ostap, E. M., Maupin, P., Doberstein, S. K., Baines, I. C., Korn, E. D. and Pollard, T. D. (2003). Dynamic localization of myosin-I to endocytic structures in Acanthamoeba. Cell Motil. Cytoskeleton (in press).
Redowicz, M. J., Martin, B., Zolkiewski, M., Ginsburg, A. and
Korn, E. D. (1994). Effects of phosphorylation and
nucleotides on the conformation of myosin II from Acanthamoeba
castellanii. J. Biol. Chem.
269,13558
-13563.
Rimm, D. L., Kaiser, D. A., Bhandari, D., Maupin, P., Kiehart, D. P., and Pollard, T. D. (1990). Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites. J. Cell Biol. 111,2405 -2416.[Abstract]
Roberson, H., Langdon, W. Y., Thien, C. B. F. and Bowtell, D. D. L. (1997). A c-Cbl yeast two hybrid screen reveals interactions with 14-3-3 isoforms and cytoskeletal components. Biochem. Biophys. Res. Commun. 240, 46-50.[CrossRef][Medline]
Sinard, J. H. and Pollard, T. D. (1989). The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II. J Cell Biol. 109,1529 -1535.[Abstract]
Sinard, J. H., Stafford, W. F. and Pollard, T. D. (1989). The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps. J. Cell Biol. 109,1537 -1547.[Abstract]
Sinard, J. H., Rimm, D. L. and Pollard, T. D. (1990). Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. II. Assembly properties of tails with NH2-and COOH-terminal deletions. J. Cell Biol. 111,2417 -2426.[Abstract]
Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M. and Borisy,
G. G. (1997). Analysis of the actin-myosin II system in fish
epidermal keratocytes: mechanism of cell body translocation. J.
Cell Biol. 139,397
-415.
Tang, N. and Ostap, E. M. (2001). Motor domain-dependent localization of myo1b (myr-1). Curr. Biol. 11,1131 -1135.[CrossRef][Medline]
Wei, Q. and Adelstein, R. S. (2000).
Conditional expression of a truncated fragment of nonmuscle myosin II-A alters
cell shape but not cytokinesis in HeLa cells. Mol. Biol.
Cell 11,3617
-3627.
Yonemura, S. and Pollard, T. D. (1992). The localization of myosin I and myosin II in Acanthamoeba by fluorescence microscopy. J. Cell Sci. 102,629 -642.[Abstract]
Yumura, S. (2001). Myosin II dynamics and
cortical flow during contractile ring formation in Dictyostelium
cells. J. Cell Biol.
154,137
-146.
Yumura, S., Mori, H. and Fukui, Y. (1984). Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence. J. Cell Biol. 99,894 -899.[Abstract]
Xu, P., Zot, A. and Zot, H. G. (1995).
Identification of Acan125 as a myosin-I-binding protein present with myosin-I
on cellular organelles of Acanthamoeba. J. Biol. Chem.
270,25316
-25319.
Xu, P., Mitchelhill, K. I., Kobe, B., Kemp, B. E. and Zot, H.
G. (1997). The myosin-I-binding protein Acan125 binds the SH3
domain and belongs to the superfamily of leucine-rich repeat proteins.
Proc. Natl. Acad. Sci. USA
94,3685
-3699.
Zang, J. H. and Spudich, J. A. (1998). Myosin
II localization during cytokinesis occurs by a mechanism that does not require
its motor domain. Proc. Natl. Acad. Sci. USA
95,13652
-13657.
Zot, H. G., Bhaskara, V. and Liu, L. (2000). Acan125 binding to the SH3 domain of Acanthamoeba myosin-IC. Arch. Biochem. Biophys. 375,161 -164.[CrossRef][Medline]