* Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and Department of Cell Biology and
Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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Abstract |
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The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a "classical" myosin-I, Acanthamoeba myosin-IB (MIB), at ~18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ~10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.
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Introduction |
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THE myosin superfamily consists of at least 12 distinct classes that vary both in the sequence of their
conserved myosin catalytic domains as well as in
their unique tails (Mooseker and Cheney, 1995; Sellers
and Goodson, 1995
). For many years the only known myosins were the double-headed, filament-forming myosins
found in muscle (conventional myosins or myosins-II).
The remaining classes of myosin have been termed "unconventional myosins" to differentiate them from the myosins-II. Probably the most thoroughly studied class of unconventional myosins is the myosin-I class. These small,
single-headed myosins bind to membrane lipids through a
basic domain in their tail (for review see Pollard et al.,
1991
; Mooseker and Cheney, 1995
). The first unconventional myosin (and first myosin-I) was isolated from Acanthamoeba castellanii (Pollard and Korn, 1973
a,b), and was
purified on the basis of its K+, EDTA, and actin-activated
MgATPase activities. However, this myosin was unusual
in that it had a single heavy chain of ~140 kD, in contrast
to the two ~200-kD heavy chains of myosin-II (Pollard and Korn, 1973
a).
Three isoforms of the classical Acanthamoeba myosins-I
are now known: myosins-IA, -IB, and -IC (Maruta and
Korn, 1977a,b; Maruta et al., 1979
). Each of the isoforms
consists of a conserved myosin catalytic domain, a binding
site for one or two light chains, a basic domain, a GPA1(Q)
domain (rich in glycine, proline and alanine [glutamine]), and an scr-homology domain-3 (SH3) domain (Pollard
et al., 1991
; Mooseker and Cheney, 1995
). These myosins-I
can associate with membranes or with actin filaments
through their tail domains. An electrostatic association of
myosin-I with anionic phospholipids and with base-stripped
membranes has been shown to occur (Adams and Pollard,
1989
; Miyata et al., 1989
; Hayden et al., 1990
), and this interaction has been mapped to the basic domain (Doberstein and Pollard, 1992
). Interestingly, these myosins also
contain a second, ATP-insensitive actin binding site (Lynch
et al., 1986
) enabling them to mediate actin-actin movements (Albanesi et al., 1985
; Fujisaki et al., 1985
). In myosin-IA (Lynch et al., 1986
) and myosin-IC (Doberstein and
Pollard, 1992
), this binding site was localized to the GPA
domain.
Acanthamoeba myosins-I have maximal steady-state actin-activated ATPase rates of ~10-20 s1 (Pollard and
Korn, 1973
b; Albanesi et al., 1983
), and an unusual triphasic dependence upon actin concentration (Pollard and
Korn, 1973
b; Albanesi et al., 1983
). This triphasic activation is due to the actin cross-linking ability imparted by the
ATP-insensitive actin binding site on the tail (Albanesi et al.,
1985
). Analysis of the individual steps in the ATPase cycle
by transient kinetics revealed that the mechanism of myosin-IA is similar to slow skeletal muscle myosin, whereas
myosin-IB (MIB) is similar to fast skeletal muscle myosin
(Ostap and Pollard, 1996
). The in vitro motility of myosin-I
has also been well characterized (Zot et al., 1992
). The
maximal rate of filament sliding is ~0.2 µm s
1. Interestingly, this rate is ~10-50× slower than the rates observed for skeletal muscle myosin, even though the ATPase rates
are comparable.
MIB consists of a 125-kD heavy chain and a single 27-kD
light chain (Maruta et al., 1979; Jung et al., 1987
). This isoform is primarily associated with the plasma membrane as
well as vacuolar membranes (Baines et al., 1992
). MIB appears to be associated with the plasma membrane at sites of
phagocytosis and was concentrated at the tips of pseudopodia (Baines et al., 1992
). This localization suggests that
MIB may be involved in membrane dynamics at the cell
surface. MIB is regulated by heavy chain phosphorylation of serine 411 (Brzeska et al., 1989
, 1990
), which is located
at the actin-binding site (Rayment et al., 1993
). Similar to
the myosin-I isoforms in Acanthamoeba, heavy chain
phosphorylation results in >20-fold activation of the actin-activated myosin-I ATPase activity (Albanesi et al., 1983
).
This activation is not the result of changes in the binding
of myosin-I to F-actin (Albanesi et al., 1983
; Ostap and
Pollard, 1996
). The transient kinetic studies of Ostap and
Pollard (1996)
suggest that phosphorylation regulates the
rate-limiting phosphate release step, the transition from
weakly bound intermediates in rapid equilibrium with actin to strongly bound states, capable of sustaining force.
Despite the extensive analysis of ameboid myosin-I biochemical properties and in vivo function, there is little
structural information on these myosins. The only detailed
structural information available for the myosins-I comes
from recent electron microscopy studies on brush border
myosin-I (BBM-I) (Jontes et al., 1995; Jontes and Milligan, 1997a
,b; Whittaker and Milligan, 1997
), a structurally
distinct myosin-I subtype. Therefore, we investigated the
structure of a "classical," ameboid-type myosin, Acanthamoeba MIB using electron microscopy. First, electron
micrographs of negatively stained two-dimensional (2D)
crystals were used to generate a projection map of MIB at
~18 Å resolution. In addition, we used cryoelectron microscopy and helical image analysis to produce a moderate
resolution three-dimensional (3D) map (30 Å) of actin filaments decorated with MIB. These studies enabled us to
compare the structure of MIB with BBM-I. The comparison of MIB with BBM-I reveals marked structural differences in the tail domains of these two proteins; MIB appears to have a much shorter "lever arm" and a more
compact tail, whereas most of the BBM-I mass is composed of an extended light chain-binding domain (LCBD). In addition, the MIB catalytic domain appears to be
slightly tilted compared to BBM-I, with respect to the
F-actin axis. Our structural results suggest that these two
types of myosin-I may have distinct intracellular functions.
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Materials and Methods |
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Protein Preparation
MIB was prepared as described by Lynch et al. (1991). For 2D crystallization experiments, the MIB was frozen in 10-µl aliquots and then stored at
80°C until use. For cryo-EM experiments, the protein was maintained
on ice and generally was used within a few days of thawing and within 2 wk
of its preparation. Actin was prepared by the method of Spudich and Watt
(1971)
.
2D Crystallization
MIB was diluted to 250 µg/ml in 10 mM Tris, pH 7.0, 13 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1mM DTT, and 5% polyethylene glycol 10,000 (final concentrations). 15-µl drops were placed in 4 × 1 mm-teflon wells and then covered with 0.5-1.0 µl of 0.5 mg/ml 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (Avanti Polar Lipids, Inc., Alabaster, AL), which had been diluted in chloroform. The protein solutions were incubated at room temperature for 8 h. Lipid films were adsorbed to carbon-coated copper grids by gently placing a grid carbon side down onto each well and then picking up the grid after 1-2 min. The grids were then negatively stained for 30-60 s in 2% uranyl acetate.
Electron Microscopy and Image Analysis of 2D Crystals
Images were collected in a transmission electron microscope (model
CM12; Philips Electron Optics, Eindhoven, The Netherlands) operating at
an accelerating voltage of 100 kV. Images were collected at a nominal
35,000× and at an underfocus of ~0.5 µm. Selected areas were scanned
on a scanning microdensitometer (model 1010GM; Perkin-Elmer Corp.,
Norwalk, CT) using a spot and step size of 20 µm, corresponding to 5.71 Å at the specimen plane. Images were processed essentially as described in
Jontes and Milligan (1997a). Lattice unbending was performed as described in Henderson et al. (1986)
. Only reflections present in three or
more of the five images were used to calculate the projection map; this
had the effect of truncating the data to a resolution of ~18 Å.
Preparation of Grids for Cryoelectron Microscopy
F-actin was diluted to ~20-40 µg/ml (~0.5-1.0 µM) in 10 mM Tris, pH 7.5, 100 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA, and then applied to electron microscopy grids covered with a holey carbon film. After ~2 min, the grids were washed with two drops of buffer and then myosin was applied to the grid at 1.0-1.5 mg/ml (6.5-10 µM). After another 2 min, the grids were blotted with filter paper and then plunged into ethane slush. Grids were stored under liquid nitrogen until use.
Cryoelectron Microscopy and Helical Image Analysis
Grids of actoMIB were mounted in a cryo stage (model 626; Gatan, Inc., Pleasanton, CA) and then inserted into a transmission electron microscope (model CM200; Philips Electron Optics) operating at an accelerating voltage of 100 kV. Images of filaments were taken under low-dose conditions at a nominal 38,000× and at an underfocus of ~1.5-2.0 µm.
Electron micrographs were screened in the optical diffractometer and
then filaments were selected for scanning and computer processing. Images were required to be free of astigmatism and drift and to have appropriate levels of defocus. Selected images were then scanned using spot and
step sizes of 25 µm, corresponding to 6.58 Å at the specimen. The PHOELIX software package (Whittaker et al., 1995; Carragher et al., 1996
) was
used for helical image processing performed essentially as described elsewhere (DeRosier and Moore, 1970
; Whittaker et al., 1995
). Steps included
straightening of the filament axis, as well as correction for the contrast
transfer function. Data were collected for Bessel orders
15 out to the 54th layer line (~27 Å).
The layer-line data from each filament were moved to a common phase
origin and then averaged. One of the individual data sets was used as a
template for the initial round of fitting and averaging. The fitting and averaging procedure was iterated four times, with the averaged data set
from the previous round acting as the new template for each successive
round. The layer-line data from the final average was then used to "sniff"
each individual data set (Morgan et al., 1995). The sniffed data were fit
and averaged twice. The effect of sniffing on the strong layer lines was
negligible but significantly improved the weaker layer lines by reducing scatter in the phases and boosting the amplitudes. The averaged layer-line
data were truncated to 30 Å and were then used in a Fourier-Bessel synthesis to produce a 3D map. 26 layer lines were used to calculate the final
map. Surface representations were made using the program SYNU
(Hessler et al., 1992
). Crystal structures were fit to the EM density using
the program O (Jones et al., 1990).
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Results |
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2D Crystals of MIB
MIB associates with monolayers of phosphatidylserine
across a wide range of conditions (Doberstein and Pollard,
1992), but only small areas (~0.2-0.3 µm on edge) were
ordered well enough for structural analysis (Fig. 1). These
2D crystals had p1 symmetry with lattice constants a = b = 56.2 ± 2.3 Å,
= 119.0 ± 1° and diffracted to ~18 Å resolution (Fig. 1 b). A projection map calculated from an average of five such images (average phase residual of 13.7 ± 3.8°) reveals a compact, globular asymmetric unit, ~56 Å × ~40 Å (Fig. 1 d). These globular densities are connected
by a smaller density, a feature that is consistently present
in each of the individual images. The strength of this connection suggests that mass from adjacent myosin molecules might be partially overlapping in projection. The low
yield of well-ordered crystals prevented us from attempting a tilt series reconstruction of this protein, as was done
for BBM-I (Jontes and Milligan, 1997a
).
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3D Map of MIB
Cryoelectron microscopy (Milligan and Flicker, 1987;
Dubochet et al., 1988
) of actin filaments decorated with
purified Acanthamoeba MIB provided additional 3D information on MIB. Images of straightened actoMIB filaments (Fig. 2) tend to have a high background like BBM-I
(Jontes and Milligan, 1997b
). This may be due to a tendency of MIB to aggregate, even at KCl concentrations of
100 mM (Ostap, E.M., unpublished observations). 32 near
or far sides from 17 filaments (4,239 actomyosin particles)
were averaged to produce the layer-line data shown in Fig. 3.
The mean phase residual for the fitting at the last iteration
was 35.7°. The data were truncated to 30 Å and then used in
a Fourier-Bessel synthesis to generate the 3D map (Fig. 4).
|
|
|
The 3D map of MIB has two distinguishable regions: a
head, which contains the catalytic domain and LCBD, and
a tail that presumably includes the lipid-binding domain,
ATP-insensitive actin-binding site, and SH3 domain (Fig.
4 b). The globular catalytic domain binds tangentially to
the actin filament in a manner similar to other myosins
(Milligan and Flicker, 1987; Jontes et al., 1995
; Whittaker
et al., 1995
). The massive MIB tail extends out from the filament axis for a short distance before angling sharply downward toward the barbed end of the filament (Fig. 4).
Contacts between the tails of adjacent MIB molecules
(Fig. 4) might be specific, although a gap may not be resolvable at 30 Å resolution.
Comparison of the MIB and BBM-I Tails
The map of MIB presented above allows a detailed structural comparison with BBM-I (Jontes et al., 1995; Jontes
and Milligan, 1997b
). Although both MIB and BBM-I are
classified as myosins-I, they represent two distinct subtypes based on comparisons of catalytic domain sequences
and of the organization of their divergent tail domains
(Fig. 5; Mooseker and Cheney, 1995
; Sellers and Goodson, 1995
). The BBM-I tail consists of a single ~30-kD carboxy-terminal domain, characterized by a large proportion
of basic amino acids. The MIB tail is more complex, having a GPA domain and an SH3 domain, in addition to the
basic lipid-binding domain. The MIB tail is much more
globular and compact than the BBM-I tail, which is a small
and elongated density present just beyond the extended
LCBD. Most of the mass of the MIB tail is also distal to
the LCBD domain and must represent the lipid-binding
region, the GPA domain, and the SH3 domain (Fig. 6).
|
|
Comparison of the MIB and BBM-I Heads
Although the tails exhibit substantial structural divergence, the motor domains of the two myosins-I are quite
similar in shape (Fig. 6). To obtain a more objective comparison, the C backbone of the skeletal muscle myosin
catalytic domain (Rayment et al., 1993
) was fit into the
EM maps to provide a visual assessment of the similarity
of the molecular envelopes of MIB and BBM-I (Fig. 7).
The crystal structure of the catalytic domain fits the 3D
map of MIB quite closely. The results of this modeling are qualitatively similar to those obtained for the fitting to
BBM-I. This indicates that, to the resolution of our EM
maps, the molecular envelopes of the MIB and BBM-I
motor domains are indistinguishable, as expected from
their sequence homology.
|
Although the molecular envelopes of the two myosin
catalytic domains are very similar, they appear to interact
with actin differently; the MIB is inclined at an angle relative to BBM-I (Fig. 6). In best fits (as determined by eye)
of the X-ray structure to the two maps (Fig. 7), MIB is pivoted about the actin binding site by ~10° toward the
barbed end of the actin. Whittaker and Milligan (1997)
have previously demonstrated that BBM-I has an attachment to actin indistinguishable from that found for skeletal muscle myosin, indicating that the interaction of MIB
with F-actin is distinct from both BBM-I and skeletal muscle subfragment-1.
The most prominent feature of the BBM-I molecule is
the large, extended LCBD. The LCBD, with three associated calmodulin light chains, accounts for most of the visible mass beyond the BBM-I catalytic domain (Jontes and
Milligan, 1997a,b). This part of the molecule projects out
orthogonally from the filament axis, placing the lipid-binding domain at high radius (Jontes and Milligan, 1997b
).
The ~30 kD lipid-binding domain of BBM-I is the most
distal density in the 3D map and extends to ~200 Å from
the actin interface, ~80 Å further than MIB. The light
chain-binding region of MIB consists of a single, 27-kD
light chain bound to a lone IQ motif positioned just distal
to the catalytic domain. This difference in the light chain-binding region constitutes one of the primary differences
between the structures of these two myosins.
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Discussion |
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The MIB Tail Domain
One of the most interesting aspects of the myosins-I is
their interaction with plasma membranes. The myosins-I
bind membranes in vitro (Adams and Pollard, 1989; Miyata
et al., 1989
; Hayden et al., 1990
; Doberstein and Pollard,
1992
) and are associated with membranes in vivo (Matsudaira and Burgess, 1979
; Baines et al., 1992
, 1995
; Fath and
Burgess, 1993
; Fath et al., 1994
). The MIB tail is particularly interesting due to its relative complexity compared to
myosin-II, as well as to BBM-I. In addition to a putative lipid-binding site, it also has a second actin-binding site
(allowing MIB to mediate actin-actin movements), as well
as an SH3 domain. At 30 Å resolution, it is not possible to
identify the distinct domains of the MIB tail, although it
seems reasonable to suppose that the lipid binding site
would be on the outer surface of the tail facing away from
the actin (Fig. 6). In this orientation, MIB would assume a
rather stubby appearance on the membrane (Fig. 8). This
may result in significant geometric contraints placed upon
the interaction of MIB with both membrane and actin. It
has also been shown that membrane-bound MIB cannot
also bind to actin through its ATP-insensitive actin-binding site (Miyata et al., 1989
; Zot et al., 1992
), suggesting
that the secondary actin-binding site is also on the outer
surface of the tail.
|
The rather compact LCBD and tail of MIB contrasts sharply with the long, extended structure of the BBM-I LCBD and tail (refer to Fig. 6). This structural difference may result in a markedly reduced working stroke for MIB compared with BBM-I or skeletal muscle myosin. The reduced size of the putative MIB lever arm may suggest that MIB moves as little as one-third the distance of BBM-I for each ATP hydrolyzed. This could explain the fact that the rates of in vitro motility for the ameboid myosins-I are much slower than would be expected based on their ATPase rates.
Jontes and Milligan (1997a,b) have suggested that the
junction between the lipid-binding domain and the third
calmodulin light chain might act like a hinge allowing
BBM-I to pivot and interact with actin that approached
the membrane from a range of angles and distances (Fig.
8). The much more massive junction in MIB makes it appear unlikely that MIB could have a similar degree of flexibility. This constraint could be reflected by the types of
activities in which these two myosins-I are involved.
The strong connection between adjacent MIB tails at
high radius in our 3D map (refer to Fig. 4), may represent
a significant protein-protein interaction or simply may be
an unresolved discontinuity. However, the projection map
calculated from 2D crystals is consistent with this interaction being "real". First, the lattice constants of the crystal
and the shape of the asymmetric unit indicate that the myosin must be sitting upright on the membrane with partial superposition of adjacent molecules. Since the MIB tail is
known to bind to phosphatidylserine, we assume that the
tail is associated with the lipid monolayer and that the
head points away from the membrane (Fig. 8). Given this
orientation and the close packing of the molecules (refer
to Fig. 1 c), it is likely that adjacent tails are in close contact. Furthermore, the spacing of 56 Å found in the planar
crystal is very similar to the ~55 Å which would separate
MIB molecules bound to successive subunits along the actin helix. Taken together, it is quite possible that the interaction observed in the helical map is also present in the 2D
crystals. If this is the case, then the helical string of MIB
molecules would have to "straighten out" to pack into the
crystal (refer to Fig. 1 c). Given the kinetic evidence that
MIB cannot work alone or in small numbers (Ostap and
Pollard, 1996), this interaction between tails may contribute to the assembly of myosins-I into small functional
groups, or "plaques". Consistent with this possibility, gold-labeled antibodies to MIB tended to cluster together on
cellular membranes (Baines et al., 1992
). Further studies
will be required to determine whether or not MIB self-
associates in vivo and what, if any, functional implications
this might have.
Interaction of the MIB and BBM-I Catalytic Domains with F-Actin
Although the tails of MIB and BBM-I differ substantially,
the catalytic domains appear very similar (refer to Figs. 6
and 7). Well-conserved proteins exhibiting similar structures at ~30 Å resolution is not surprising. In spite of the
overall similarity in shape of the catalytic domains, we observe an intriguing difference in the angles of attachment
to actin. The MIB molecule is inclined by ~10° at the actin-binding site relative to BBM-I (refer to Figs. 6 and 7).
Interactions of the tails of adjacent myosins may contribute to this difference, although there is little evidence to
suggest that a myosin bound in rigor would be flexible at
the site of actin attachment (Cooke, 1981).
The absence of phosphorlyation of the MIB heavy chain
may contribute to the difference in attachment. Phosphorylation of serine 411, located at the actin-binding site, activates the MIB enzymatic and motile activities (Brzeska et
al., 1989; 1990
). The MIB used in this study was unphosphorylated and, therefore, inactive. In contrast to the ameboid myosin-Is, most members of the myosin superfamily
have an acidic residue at this site, the TEDS rule site, and
can be considered to be constitutively phosphorylated and
active (Bement and Mooseker, 1995
; Mooseker and Cheney,
1995
). If this idea is true, the attachment of phosphorylated MIB to actin may more closely resemble that of
BBM-I. Alternatively, the altered interaction of MIB with
actin might reflect a functional requirement unique to this
myosin subclass. As mentioned above, the stubby appearance of MIB on the membrane may place geometric constraints on the interaction of MIB with actin, and then the altered attachment may represent a form of compensation
or accommodation. It may also be worth noting that the
actin used in this study (purified from rabbit back muscle)
differs slightly from the actin with which either of these
myosins would interact in vivo. Finally, the difference in
angle of the attachment of MIB or BBM-I to actin might
simply be due to an inherent difference between these two divergent motors, lacking any significant functional consequences.
Implications for Myosin-I Function
Structural and biochemical analysis of the Acanthamoeba
myosin-Is and BBM-I have revealed major differences in
the properties of these two types of myosin-I. Myosins-IA
and -IB have kinetic properties very similar to those of the
conventional myosins (Ostap and Pollard, 1996), having
relatively robust rates of actin-activated ATP hydrolysis
and spending a relatively short percentage of their cycles
attached to actin. BBM-I, by contrast, has a very slow ATPase (Collins et al., 1990
; Wolenski et al., 1993
; Jontes et al., 1997
) and may spend a longer time attached to actin
(Jontes et al., 1997
). Additionally, our structural results
suggest that MIB likely has a much smaller working stroke
than BBM-I, and that MIB may have the tendency to self-associate. Taken together, the evidence suggests that these
myosins serve different subsets of cellular functions. Consistent with their kinetics and intracellular locations, the
ameboid myosins-I may act as dynamic linkages in the cell
periphery, forming contractile networks in the cell cortex and under the plasma membrane. These myosins-I would
function as a cellular population and would be able to
modulate and coordinate many cell surface activities such
as exocytosis, endocytosis, cell migration, and pseudopod
and filopodial extension (Jung et al., 1993
, 1996
; Novak et
al., 1995
; Novak and Titus, 1997
). The BBM-I type of myosin-I would be better suited to motile activites where they
worked in smaller numbers, such as transporting vesicles (Drenkhahn and Dermietzel, 1988
; Fath and Burgess,
1993
; Fath et al., 1994
) or acting as an adaptation motor
(Assad and Corey, 1992
; Hudspeth and Gillespie, 1993).
Thus, analysis of the biochemical and structural properties
of unconventional myosins may help to focus investigation
into their cellular functions.
![]() |
Footnotes |
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Received for publication 14 November 1997 and in revised form 26 January 1998.
J.D. Jontes would like to thank B. Carragher (Beckman Institute, University of Illinois, Urbana-Champaign, IL) for access to the Philips CM200TEM and for her hospitality during collection of the MIB helical data.This work was supported by research grants from the National Institutes of Health (Bethesda, MD) to R.A. Milligan (AR-39155), and to T.D. Pollard (GM-26132). E.M. Ostap was supported in part by a grant from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (DRG-1294). J.D. Jontes was supported by a predoctoral fellowship from the Howard Hughes Medical Institute (Chevy Chase, MD).
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Abbreviations used in this paper |
---|
2D, two-dimensional; 3D, three- dimensional; BBM-I, brush border myosin-I; GPA, glycine-proline-alanine; LCBD, light chain-binding domain; MIB, Acanthamoeba myosin-IB; SH3, src-homology domain-3.
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References |
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