Cardiomyopathies are a major health problem, with inherited cardiomyopathies, many of which are caused by mutations in genes encoding sarcomeric proteins, constituting an ever-increasing fraction of cases. To begin to study the mechanisms by which these mutations cause disease, we have employed an integrative modelling approach to study the interactions between tropomyosin and actin. Starting from the existing blocked state model, we identified a specific zone on the actin surface which is highly favourable to support tropomyosin sliding from the blocked/closed states to the open state. We then analysed the predicted actin-tropomyosin interface regions for the three states. Each quasi-repeat of tropomyosin was studied for its interaction strength and evolutionary conservation to focus on smaller surface zones. Finally, we show that the distribution of the known cardiomyopathy mutations of α-tropomyosin is consistent with our model. This analysis provides structural insights into the possible mode of interactions between tropomyosin and actin in the open state for the first time.
Video Abstract Available from http://la-press.com/t.php?i=9798
cardiomyopathy mutations; cardiac thin filament; modeling; tropomyosin; period 2
Myosins have diverse mechanical properties reflecting a range of cellular roles. A major challenge is to understand the structural basis for generating novel functions from a common motor core. Myosin VI is specialized for processive motion toward the (-) end of actin filaments. We have used engineered myosin VI motors to test and refine the “redirected power stroke” model for (-) end directionality, and to explore poorly understood structural requirements for processive stepping. Guided by crystal structures and molecular modeling, we fused artificial lever arms to the catalytic head of myosin VI at several positions, retaining varying amounts of native structure. We found that an 18 residue alpha-helical insert is sufficient to reverse the directionality of the motor, with no requirement for any calmodulin light chains. Further, we observed robust processive stepping of motors with artificial lever arms, demonstrating that processivity can arise without optimizing lever arm composition or mechanics.
We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron Paramagnetic Resonance spectroscopy reveals that the pocket is in an equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH and ΔS, even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket, and the observation that fast myosins are less efficient than slow myosins.
The myosin superfamily is a versatile group of molecular motors involved in the transport of specific biomolecules, vesicles and organelles in eukaryotic cells. The processivity of myosins along an actin filament and transport of intracellular ‘cargo’ are achieved by generating physical force from chemical energy of ATP followed by appropriate conformational changes. The typical myosin has a head domain, which harbors an ATP binding site, an actin binding site, and a light-chain bound ‘lever arm’, followed often by a coiled coil domain and a cargo binding domain. Evolution of myosins started at the point of evolution of eukaryotes, S. cerevisiae being the simplest one known to contain these molecular motors. The coiled coil domain of the myosin classes II, V and VI in whole genomes of several model organisms display differences in the length and the strength of interactions at the coiled coil interface. Myosin II sequences have long-length coiled coil regions that are predicted to have a highly stable dimeric interface. These are interrupted, however, by regions that are predicted to be unstable, indicating possibilities of alternate conformations, associations to make thick filaments, and interactions with other molecules. Myosin V sequences retain intermittent regions of strong and weak interactions, whereas myosin VI sequences are relatively devoid of strong coiled coil motifs. Structural deviations at coiled coil regions could be important for carrying out normal biological function of these proteins.
myosin structure; myosin domain architecture; coiled coil
A mere forty years ago it was unclear what motor molecules exist in cells that could be responsible for the variety of nonmuscle cell movements, including the “saltatory cytoplasmic particle movements” apparent by light microscopy. One wondered whether nonmuscle cells might have a myosin-like molecule, well known to investigators of muscle. Now we know that there are more than a hundred different molecular motors in eukaryotic cells that drive numerous biological processes and organize the cell's dynamic city plan. Furthermore, in vitro motility assays, taken to the single-molecule level using techniques of physics, have allowed detailed characterization of the processes by which motor molecules transduce the chemical energy of ATP hydrolysis into mechanical movement. Molecular motor research is now at an exciting threshold of being able to enter into the realm of clinical applications.
Myosin VI is a molecular motor that is thought to function as both a transporter and a cytoskeletal anchor in vivo. Here we use optical tweezers to examine force generation by single molecules of myosin VI under physiological nucleotide concentrations. We find that myosin VI is an efficient transporter at loads up to ~2 pN but acts as a cytoskeletal anchor at higher loads. Our data and resulting model are consistent with an indirect coupling of global structural motions to nucleotide binding and release. The model provides a mechanism by which load may regulate the dual functions of myosin VI in vivo. Our results suggest that myosin VI kinetics are tuned such that the motor maintains a consistent level of mechanical tension within the cell, a property potentially shared by other mechanosensitive proteins.
single molecule biophysics; optical tweezers; molecular motors; mechanical force; cytoskeleton
We optimally localize isolated fluorescent beads and molecules imaged as diffraction-limited spots, determine the orientation of molecules, and present reliable formulae for the precisions of various localization methods. For beads, theory and experimental data both show that unweighted least-squares fitting of a Gaussian squanders one third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, while maximum likelihood fitting is practically optimal.
Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator [1, 2]. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear.
By analyzing spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments, and the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages; 1) turnover of thick filaments throughout the cell cycle, 2) MHC-based control of myosin assembly at the metaphase-anaphase transition, and 3) redistribution/activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering pre-assembled filaments to the equatorial zone via cortical flow.
Multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.
cytokinesis; myosin II; myosin regulatory light chain; FRAP; Drosophila S2 cell
The authors propose a new mechanism for actin-based force generation based on results using chains of actin-grafted magnetic colloids.
The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.
Actin self-assembles into filaments, and this produces forces that deform cell membranes in a large number of motile processes. While physical measurements have been performed of the force produced by growth of either a single filament or a large intricate array of filaments organized in an active macroscopic gel, these measurements don't provide a clear picture of how force is produced by the assembly of each filament within a complex structure. The present study explores a situation between these two extremes by measuring the force produced by the assembly of a small number of filaments. We developed a method in which actin filaments grow from the surface of magnetic beads that are aligned by a controlled magnetic field. The distance between beads in a chain-like arrangement increases with time when the force is kept constant. We observe that the growth of actin filaments is not affected by the load, in contrast to the widely accepted “Brownian ratchet model.” Instead, our results suggest that the surface opposite growing filaments imposes restrictions on the rotational fluctuations of a filament at its free hinge anchoring point, inducing a repulsive force. The confinement of filaments increases as they grow, and this in turn increases the repulsive force developed by their growth. This entropy-based mechanism may operate during motile processes when actin networks are loosely organized.
Myosins are ATP-driven linear molecular motors that work as cellular force
generators, transporters, and force sensors. These functions are driven by
large-scale nucleotide-dependent conformational changes, termed
“strokes”; the “power stroke” is the force-generating
swinging of the myosin light chain–binding “neck” domain
relative to the motor domain “head” while bound to actin; the
“recovery stroke” is the necessary initial motion that primes, or
“cocks,” myosin while detached from actin. Myosin Va is a processive
dimer that steps unidirectionally along actin following a “hand over
hand” mechanism in which the trailing head detaches and steps forward
∼72 nm. Despite large rotational Brownian motion of the detached head about
a free joint adjoining the two necks, unidirectional stepping is achieved, in
part by the power stroke of the attached head that moves the joint forward.
However, the power stroke alone cannot fully account for preferential forward
site binding since the orientation and angle stability of the detached head,
which is determined by the properties of the recovery stroke, dictate actin
binding site accessibility. Here, we directly observe the recovery stroke
dynamics and fluctuations of myosin Va using a novel, transient caged
ATP-controlling system that maintains constant ATP levels through stepwise
UV-pulse sequences of varying intensity. We immobilized the neck of monomeric
myosin Va on a surface and observed real time motions of bead(s) attached
site-specifically to the head. ATP induces a transient swing of the neck to the
post-recovery stroke conformation, where it remains for ∼40 s, until ATP
hydrolysis products are released. Angle distributions indicate that the
post-recovery stroke conformation is stabilized by ≥5
kBT of energy. The high kinetic
and energetic stability of the post-recovery stroke conformation favors
preferential binding of the detached head to a forward site 72 nm away. Thus,
the recovery stroke contributes to unidirectional stepping of myosin Va.
Myosin Va is a “two-legged” ATP-dependent linear molecular motor that
transports cellular organelles by “stepping” along actin filaments
in a processive manner analogous to human walking, the two “feet”
alternating between forward and backward positions. During stepping, the lifted
leg undergoes rotational Brownian movements around a free joint at the
leg–leg junction. Although these movements are random, the lifted foot
lands preferentially on forward sites and rarely steps backward. This
directional bias arises in part from the forward movement of the junction
bending the “ankle” of the attached leg. Here, we show that the
lifted foot also plays a role in the direction of stepping by controlling the
orientation of its actin-binding site (the “sole”), which dictates
the accessibility of potential stepping positions. We observed the ATP-dependent
foot orientation and its stabilizing on individual myosin Va molecules in real
time under an optical microscope; we show that the lifted foot of walking myosin
Va is oriented in a “toe-down” conformation so that binding to a
forward site on actin is preferred largely over backward or adjacent sites.
Thus, the great kinetic and energetic stability of the myosin Va lifted foot
conformation contributes to unidirectional stepping along actin filaments.
The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual ~ 180° lever arm swing and achieves its large step size using special structural features in its tail domain.
A combination of experimentation and modeling reveal that multiple myosin VI molecules coordinately transport cargo over the actin filament network.
Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.
Multiple mitotic motors coordinate their signals to ensure that the actomyosin contractile ring forms in the right place during cytokinesis.
Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.
Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take ~36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single α-helix of ~10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the ~36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.
We present a computational method that predicts a pathway of residues that mediate protein allosteric communication. The pathway is predicted using only a combination of distance constraints between contiguous residues and evolutionary data. We applied this analysis to find pathways of conserved residues connecting the myosin ATP binding site to the lever arm. These pathway residues may mediate the allosteric communication that couples ATP hydrolysis to the lever arm recovery stroke. Having examined pre-stroke conformations of Dictyostelium, scallop, and chicken myosin II as well as Dictyostelium myosin I, we observed a conserved pathway traversing switch II and the relay helix, which is consistent with the understood need for allosteric communication in this conformation. We also examined post-rigor and rigor conformations across several myosin species. Although initial residues of these paths are more heterogeneous, all but one of these paths traverse a consistent set of relay helix residues to reach the beginning of the lever arm. We discuss our results in the context of structural elements and reported mutational experiments, which substantiate the significance of the pre-stroke pathways. Our method provides a simple, computationally efficient means of predicting a set of residues that mediate allosteric communication. We provide a refined, downloadable application and source code on https://simtk.org to share this tool with the wider community (https://simtk.org/home/allopathfinder).
allostery; myosin; pathway; conservation; computation
The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6–related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.
Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.
Myosin VI is a molecular motor implicated in diverse cell processes, including trafficking endocytic vesicles into the cell, transporting proteins to the leading edge of a migrating cell, and anchoring stereocilia to the hair cells of inner ear sensory epithelia. The motor has been studied in both a monomeric and dimeric form in vitro and is reported to exist as a monomer in the cytoplasm of cells. Because the functional characteristics of the motor are dramatically different for these two forms, an understanding of the activity of myosin VI requires an understanding of its functional form in vivo. To probe the role of myosin VI in vesicle trafficking, we labeled myosin VI truncations with a fluorescent protein and studied the positioning of these constructs on endocytic vesicles. We observed nonradiative transfer of energy between the fluorescent proteins, a process that can only occur if they are brought extremely close together. Our results indicate that, when myosin VI heavy chains bind to endocytic vesicles, they are precisely positioned very close together. Work from other laboratories indicates that myosin VI heavy chains brought together in this manner are capable of dimerization. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of myosin VI's known trafficking function.
Fluorescence anisotropy measurements are used to demonstrate that, on endocytic vesicles, myosin VI heavy chains are brought together in an orientation consistent with a processive dimer, capable of its known trafficking function.
Myosin II recruitment to the equatorial cortex is one of the earliest events in establishment of the cytokinetic contractile ring. In Drosophila S2 cells, we previously showed that myosin II is recruited to the furrow independently of F-actin, and that Rho1 and Rok are essential for this recruitment . Rok phosphorylates several cellular proteins, including the myosin regulatory light chain (RLC).
Here we express phosphorylation state mimic constructs of the RLC in S2 cells to examine the role of RLC phosphorylation involving Rok in the localization of myosin. Phosphorylation of the RLC is required for myosin localization to the equatorial cortex during mitosis, and the essential role of Rok in this localization and for cytokinesis is to maintain phosphorylation of the RLC. The ability to regulate the RLC phosphorylation state spatio-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis is not phosphorylation of the RLC.
We conclude that the Rho1 pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC.
Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.
Biochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division.
MHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses.
During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring.
The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex.
The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.
We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.
cytoskeleton; cortexillin; cell cortex; cell protrusion; Rac small GTPase
Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3×Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3×Asp myosin II fails to rescue myosin II–null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II–null cells containing 3×Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled–coil structure.
myosin II; thick filament assembly; Dictyostelium; phosphorylation; suppressor screen
We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.
Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.
To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.