The biological activity of subfragment 1 prepared from heavy meromyosin

1. The action of trypsin, chymotrypsin and subtilisin on the adenosine-triphosphatase and actin-combining activities, as measured by viscometric means, of H-meromyosin were compared. 2. Subfragment 1 produced by prolonged tryptic digestion has a molecular weight of 129000. 3. The preparations isolated by gel filtration and actin combination were shown to be similar. 4. Subfragment-1 preparations possess appreciably higher adenosine-triphosphatase activities than H-meromyosin when related to total nitrogen. 5. Chromatographic and gelfiltration studies indicated that adenosine-triphosphatase activity is not distributed uniformly in all fractions of subfragment 1. 6. The Ca(2+)-activated adenosine triphosphatase of subfragment 1 was stimulated by thiol reagents in a similar fashion to myosin and H-meromyosin. 7. Subfragment 1 differed from myosin and H-meromyosin in that its adenosine triphosphatase was only slightly activated by Mg(2+) in the presence of actin. 8. A subfragment-1-like component was obtained by chymotryptic digestion of H-meromyosin. 9. The results obtained from enzymic and hydrodynamic studies and from amino acid analyses are compatible with the concept of one molecule of H-meromyosin giving rise to one molecule of subfragment 1 on proteolytic digestion.     

Desensitization of substrate inhibition of acto-H-meromyosin ATPase by treatment of H-meromyosin with rho-chloromercuribenzoate. Relation between the extent of desensitization and the amount of bound rho-chloromercuribenzoate1.

H-Meromyosin (CMB leads to betaME-H-meromyosin) was prepared by tryptic digestion of myosin, which had been treated with CMB bound to H-meromyosin and the extent of desensitization of the substrate inhibition of acto-H-meromyosin ATPase [EC 3.6.1.3.] was investigated. Both the dissociation of acto-H-meromyosin induced by ATP and substrate inhibition decreased with increase in the amount of bound CMB to a minimum value at about 1 mole of CMB bound per mole of H-meromyosin. The substrate inhibition of acto-H-meromyosin ATPase was restored to the original level by complete removal of the bound CMB by further treatment of CMB leads to beta ME-H-meromyosin with a large excess of beta-mercaptoethanol. The dissociation constant of acto-H-meromyosin in the presence of ATP decreased markedly on modification with CMB, while the maximum ATPase activity ar a sufficiently high concentration of F-actin remained essentially unchanged. Acto-H-meromyosin was reconstituted from F-actin and CMB LEADS TO beta ME-H-meromyosin, containing less than the stoichiometric amount of bound CMB. Its ATPase activity and the extent of dissociation of acto-H-meromyosin induced by ATP were explained as those of a mixture of unmodified H-meromyosin and CMB leads to beta ME-H-meromyosin containing 1 mole of CMB per mole of H-meromyosin. Half of the light chains (g2), with a molecular weight of 18,000, were removed from myosin by treatment with CMB and beta-mercaptoethanol. After this treatment, on further incubation of the myosin with a large excess of beta-mercaptoethanol, the myosin contained only half of the g2, but the substrate inhibition of acto-H-meromyosin ATPase was restored completely. The initial burst of P1 liberation and the EDTA-ATPase activity decreased to almost zero on specific modification of the SH1-groups with NEM, while the initial burst decreased to some extent and the EDTA-ATPase activity to 50% of the original value on binding of 1 mole CMB per mole of H-meromyosin. The actomyosin-type of ATPase activity was strongly inhibited by modification with CMB. The extent of the dissociation of acto-H-meromyosin induced by ATP was unaffected by modification with NEM, while it decreased on further treatment of NEM-myosin with CMB FOLLOWED BY BETA-MERCAPTOETHANOL.     

Effects of tropomyosin, troponin and Ca on the interaction between F-actin and heavy meromyosin

1. In the absence of ATP, H-meromyosin (heavy meromyosin) bound with the F-actin-tropomyosin-troponin complex up to the molar ratio of H-meromyosin to actin of 1:1, independently of the concentration of Ca²⁺.

2. In the presence of free Ca²⁺ above about 1 μM, with an increasing amount of H-meromyosin bound to a fixed amount of the F-actin-tropomyosin-troponin complex, the degree of flow birefringence decreased and the extinction angle increased. The minimum value of the birefringence and the maximum value of the extinction angle were found at the molar ratio of H-meromyosin to actin of 1:2. A further increase of bound H-meromyosin to actin restored both the degree of birefringence and the extinction angle to nearly the same level as the F-actin-tropomyosin-troponin complex only. In the absence of free Ca²⁺, the birefringence did not change with the binding of H-meromyosin.

3. This sensitivity of birefringence to the concentration of Ca²⁺ appeared only in the presence of tropomyosin and troponin. At a fixed ratio of H-meromyosin, actin and tropomyosin, the birefringence in the absence of Ca²⁺ increased with increasing amount of added troponin up to the weight ratio of troponin to actin of 1:6; whereas the birefringence in the presence of Ca²⁺ did not change.

4. At a fixed ratio of H-meromyosin to actin, the birefringence changed with increasing amount of tropomyosin added up to the weight ratio of tropomyosin to actin of 1:6; above this ratio, the birefringence was constant.

5. Subfragment S-1, prepared by the chymotryptic digestion of myosin, bound to F-actin, but the birefringence did not change even in the presence of tropomyosin and troponin.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex.

To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va-associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein-myosin Va tail construct. Myosin Va-associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.     

Local migration of myosin in F-actin plus ATP solution on the boundary of a diffusion cell.

The diffusion phenomena of myosin (myosin A, H-meromyosin or subfragment-1) in F-actin plus ATP solutions were investigated. The upper part of the diffusion cell was filled with F-actin plus ATP, and the lower part was filled with F-actin, ATP, and myosin, then both parts were brought into contact so that a boundary of the two solutions was formed and the diffusion of myosin in F-actin plus ATP solutions started. The diffusion pattern was observed with a schlieren lens system. When almost all the ATP in the lower part of the cell had been consumed by actomyosin, a hyper-sharp schlieren pattern appeared near the boundary. On analyzing this pattern, it was found that a local fast migration of proteins was occurring. Simple Brownian motion of myosin molecules could not explain the hyper-sharp phenomenon. This phenomenon occurred in ther pesence of Mg2+ or Ca2+, but very little in the presence of EDTA. Although it is well known that the superprecipitation of myosin B suspension occurs only at physiological ionic strength, this phenomenon occurred over a relatively wide range of ionic strengths. The molecular mechanism of this phenomenon is discussed in relation to the basic mechanism of the interaction between myosin and F-actin.     

SOME ASPECTS OF THE STRUCTURAL ORGANIZATION OF THE MYOFIBRIL AS REVEALED BY ANTIBODY-STAINING METHODS

From observations of fluorescent antibody staining and antibody staining in electron microscopy, evidence is presented for the following: (a) Direct contact of the actin and myosin filaments occurs at all stages of contraction. This results in inhibition of antibody staining of the H-meromyosin portion of the myosin molecule in the region of overlap of the thin and thick filaments. (b) Small structural changes occur in the thick filaments during contraction. This leads to exposure of antigenic sites of the L-meromyosin portion of the myosin molecule. The accessibility of these antigenic sites is dependent upon the sarcomere length. (c) The M line is composed of a protein which is weakly bound to the center of the thick filament and is not actin, myosin, or tropomyosin. (d) Tropomyosin as well as actin is present in the I band. (e) If actin or tropomyosin is present in the Z line, it is masked and unavailable for staining with antibody.     

Diffusion of H-meromyosin in F-actin plus ATP solution at a very low electrolyte concentration

The translational diffusion coefficient (D) of H-meromyosin in actin (F-actin) and ATP solution was measured under conditions wherein the actin-activated ATPase activity is close to its maximal value at a very low electrolyte concentration. The results were compared with similar data obtained with 0.1 M KCl, where H-meromyosin and actin were almost completely dissociated. With 0.1 M KCl, it was found that there was no dependence of the D of H-meromyosin on actin concentration. On the other hand, at a very low electrolyte concentration, it was found that the D of H-meromyosin did depend on actin concentration; at a rather high actin concentration (and activation of ATPase), it was slightly larger than at low or zero actin concentrations. This behavior of D at a low electrolyte concentration is interpreted on the assumption that even in solution, H-meromyosin molecules can actively slide on actin filaments due to the ATPase activity.     

Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat

Antitubulin, phalloidin, and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell. A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.     

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.     

The cortical microfilament system of lymphoblasts displays a periodic oscillatory activity in the absence of microtubules: implications for cell polarity

For an understanding of the role of microtubules in the definition of cell polarity, we have studied the cell surface motility of human lymphoblasts (KE37 cell line) using video microscopy, time-lapse photography, and immunofluorescent localization of F-actin and myosin. Polarized cell surface motility occurs in association with a constriction ring which forms on the centrosome side of the cell: the cytoplasm flows from the ring zone towards membrane veils which keep protruding in the same general direction. This association is ensured by microtubules: in their absence the ring is conspicuous and moves periodically back and forth across the cell, while a protrusion of membrane occurs alternately at each end of the cell when the ring is at the other. This oscillatory activity is correlated with a striking redistribution of myosin towards a cortical localization and appears to be due to the alternate flow of cortical myosin associated with the ring and to the periodic assembly of actin coupled with membrane protrusion. The ring cycle involves the progressive recruitment of myosin from a polar accumulation, or cap, its transportation across the cell and its accumulation in a new cap at the other end of the cell, suggesting an assembly-disassembly process. Inhibition of actin assembly induces, on the other hand, a dramatic microtubule-dependent cell elongation with definite polarity, likely to involve the interaction of microtubules with the cell cortex. We conclude that the polarized cell surface motility in KE37 cells is based on the periodic oscillatory activity of the actin system: a myosin-powered equatorial contraction and an actin-based membrane protrusion are concerted at the cell level and occur at opposite ends of the cell in absence of microtubules. This defines a polarity which reverses periodically as the ring moves across the cell. Microtubules impose a stable cell polarity by suppressing the ring movement. A permanent association of the myosin-powered contraction and the membrane protrusion is established which results in the unidirectional activity of the actin system. Microtubules exert their effect by controlling the recruitment of cytoplasmic myosin into the cortex, probably through their direct interaction with the cortical microfilament system.     

Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.     

Kinesin and myosin ATPases: variations on a theme.

The enzymes kinesin and myosin are examples of molecular motors which couple ATP hydrolysis to directed movement of biological structures. Myosin has been extensively studied and its structure and mechanism of coupling are known in detail. Much less is known about kinesin, but many of its major properties are similar to those of myosin. Both enzymes have two catalytic head groups at the end of a long alpha-helical rod. The head groups contain the sites for ATP hydrolysis and interaction with their respective partners for movement (microtubules or F-actin). In each case the binding and hydrolysis of ATP is rapid and the steady state ATPase rate is limited by a slow step in the region of product release. This slow release of product is accelerated by interaction with actin or microtubules coupled to changes in binding affinity. As there is no evidence for a close evolutionary link between kinesin and myosin, these and other similarities may represent convergence to set of common functional properties which are constrained by the requirements of protein structure and the use of ATP hydrolysis as a source of energy. It will be of particular interest to determine if these common properties are also shared by the large number of divergent proteins which have recently been discovered to possess a domain which is homologous to the head group of kinesin.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis.     

Zebrafish melanophilin facilitates melanosome dispersion by regulating dynein

BACKGROUND: Fish melanocytes aggregate or disperse their melanosomes in response to the level of intracellular cAMP. The role of cAMP is to regulate both melanosome travel along microtubules and their transfer between microtubules and actin. The factors that are downstream of cAMP and that directly modulate the motors responsible for melanosome transport are not known. To identify these factors, we are characterizing melanosome transport mutants in zebrafish. RESULTS: We report that a mutation (allele j120) in the gene encoding zebrafish melanophilin (Mlpha) interferes with melanosome dispersion downstream of cAMP. Based on mouse genetics, the current model of melanophilin function is that melanophilin links myosin V to melanosomes. The residues responsible for this function are conserved in the zebrafish ortholog. However, if linking myosin V to melanosomes was Mlpha's sole function, elevated cAMP would cause mlpha(j120) mutant melanocytes to hyperdisperse their melanosomes. Yet this is not what we observe. Instead, mutant melanocytes disperse their melanosomes much more slowly than normal and less than halfway to the cell margin. This defect is caused by a failure to suppress minus-end (dynein) motility along microtubules, as shown by tracking individual melanosomes. Disrupting the actin cytoskeleton, which causes wild-type melanocytes to hyperdisperse their melanosomes, does not affect dispersion in mutant melanocytes. Therefore, Mlpha regulates dynein independently of its putative linkage to myosin V. CONCLUSIONS: We propose that cAMP-induced melanosome dispersion depends on the actin-independent suppression of dynein by Mlpha and that Mlpha coordinates the early outward movement of melanosomes along microtubules and their later transfer to actin filaments.     

Noncentrosomal microtubules and type II myosins potentiate epidermal cell adhesion and barrier formation

During differentiation, many cells reorganize their microtubule cytoskeleton into noncentrosomal arrays. Although these microtubules are likely organized to meet the physiological roles of their tissues, their functions in most cell types remain unexplored. In the epidermis, differentiation induces the reorganization of microtubules to cell–cell junctions in a desmosome-dependent manner. Here, we recapitulate the reorganization of microtubules in cultured epidermal cells. Using this reorganization assay, we show that cortical microtubules recruit myosin II to the cell cortex in order to engage adherens junctions, resulting in an increase in mechanical integrity of the cell sheets. Cortical microtubules and engaged adherens junctions, in turn, increase tight junction function. In vivo, disruption of microtubules or loss of myosin IIA and B resulted in loss of tight junction–mediated barrier activity. We propose that noncentrosomal microtubules act through myosin II recruitment to potentiate cell adhesion in the differentiating epidermis, thus forming a robust mechanical and chemical barrier against the external environment.     

Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions

Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.     

Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division

We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed.