Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.
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Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment et al., 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987; Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild-type myosin, but the filaments then exhibit abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.Myosin X(Myo X) is an unconventional myosin with a tail of homology 4-band 4.1/ezrin/radixin/moesin(MyTH4-FERM) tail.It is ubiquitously expressed in various mammalian tissues.Recently,Myo X was found to interact with several molecules involved in signal transduction pathways that affected the cellular motility processes ranging from filopodial formation to cytokinesis.Myo X,as one of the MyTH-FERM myosins,is increasingly recognized as a key mediator of membrane-cytoskeleton interactions,yet the exact mechanism of its function was not fully understood.In this review,the literature on Myo X and cell motility are summarized.
Moesin
Radixin
FERM domain
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ABSTRACT Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane, while myosin MYO2 functions in actomyosin ring contraction to promote cytokinesis. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. Myl5 localized to the mitotic spindle poles and spindle microtubules during early mitosis, an area overlapping with MYO10 localization. Depletion of Myl5 led to defects in chromosome congression and to a slower progression through mitosis. We propose that Myl5 is a novel myosin RLC that is important for cell division.
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Astral microtubules
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Septin
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Abstract In the simple amoeba Dictyostelium discoideum , myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G‐protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP‐myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP‐myosin fusions, and with latrunculin A–treated cells, demonstrated that F‐actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A “headless” GFP‐myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor‐based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant‐stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant‐regulated myosin II filament assembly and translocation. Cell Motil. Cytoskeleton 53:177–188, 2002. © 2002 Wiley‐Liss, Inc.
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Septin
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Actin remodeling
Fascin
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Changes in the actin cytoskeleton associated with cell motility are attributed in part to the Rho family of GTP-binding proteins and their modulation of the PAK family of serine-threonine kinases. Chung and Firtel have identified a putative Pak (domain structure similar to mammalian Pak) in Dictyostelium discoideum that appears to regulate cytokinesis as well cell movement. Pak-null cells did not display normal polarized distribution of actin and exhibited defects in chemotaxis as well as in developmental aggregation. These cells also failed to undergo cytokinesis. Although Pak colocalized with myosin II in the cleavage furrow of dividing wild type cells, myosin II was not a substrate of Pak. However, the cytokinesis defect resembled the phenotype of myosin II-deficient cells. The authors hypothesize that Pak could inhibit myosin II heavy chain kinase, an enzyme that regulates the assembly of myosin II filaments. It remains to be determined whether this Pak is regulated by Rho proteins. Chung, C.Y., and Firtel, R.A. (1999) PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis. J. Cell Biol. 147 : 559-575. [Abstract] [Full Text]
Cleavage furrow
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Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.
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During spermatogenesis, interconnected haploid spermatids segregate undesired cellular contents into residual bodies (RBs) before detaching from RBs. It is unclear how this differentiation process is controlled to produce individual spermatids or motile spermatozoa. Here, we developed a live imaging system to visualize and investigate this process in C. elegans. We found that non-muscle myosin 2 (NMY-2)/myosin II drives incomplete cytokinesis to generate connected haploid spermatids, which are then polarized to segregate undesired cellular contents into RBs under the control of myosin II and myosin VI. NMY-2/myosin II extends from the pseudo-cleavage furrow formed between two haploid spermatids to the spermatid poles, thus promoting RB expansion. In the meantime, defective spermatogenesis 15 (SPE-15)/myosin VI migrates from spermatids towards the expanding RB to promote spermatid budding. Loss of myosin II or myosin VI causes distinct cytoplasm segregation defects, while loss of both myosins completely blocks RB formation. We found that the final separation of spermatids from RBs is achieved through myosin VI–mediated cytokinesis, while myosin II is dispensable at this step. SPE-15/myosin VI and F-actin form a detergent-resistant actomyosin VI ring that undergoes continuous contraction to promote membrane constriction between spermatid and RB. We further identified that RGS-GAIP-interacting protein C terminus (GIPC)-1 and GIPC-2 cooperate with myosin VI to regulate contractile ring formation and spermatid release. Our study reveals distinct roles of myosin II and myosin VI in spermatid differentiation and uncovers a novel myosin VI–mediated cytokinesis process that controls spermatid release.
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Cleavage furrow
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