scholarly journals Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

2015 ◽  
Vol 26 (7) ◽  
pp. 1211-1224 ◽  
Author(s):  
Zhonghui Feng ◽  
Satoshi Okada ◽  
Guoping Cai ◽  
Bing Zhou ◽  
Erfei Bi
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Membrane Trafficking ◽  
Myosin Ii ◽  
Myosin V ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Essential Light Chain ◽  
Division Site ◽  
Yeast Mutants

MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species.

scholarly journals Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast

2013 ◽  
Vol 200 (3) ◽  
pp. 271-286 ◽  
Author(s):  
Carsten Wloka ◽  
Elizabeth A. Vallen ◽  
Lydia Thé ◽  
Xiaodong Fang ◽  
Younghoon Oh ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Budding Yeast ◽  
Myosin Ii ◽  
Small Region ◽  
Myosin V ◽  
Actin Ring ◽  
Essential Light Chain ◽  
Division Site ◽  
Ring Assembly ◽  
Core Components

Core components of cytokinesis are conserved from yeast to human, but how these components are assembled into a robust machine that drives cytokinesis remains poorly understood. In this paper, we show by fluorescence recovery after photobleaching analysis that Myo1, the sole myosin-II in budding yeast, was mobile at the division site before anaphase and became immobilized shortly before cytokinesis. This immobility was independent of actin filaments or the motor domain of Myo1 but required a small region in the Myo1 tail that is thought to be involved in higher-order assembly. As expected, proteins involved in actin ring assembly (tropomyosin and formin) and membrane trafficking (myosin-V and exocyst) were dynamic during cytokinesis. Strikingly, proteins involved in septum formation (the chitin synthase Chs2) and/or its coordination with the actomyosin ring (essential light chain, IQGAP, F-BAR, etc.) displayed Myo1-dependent immobility during cytokinesis, suggesting that Myo1 plays a scaffolding role in the assembly of a cytokinesis machine.

Essential light chain of Drosophila nonmuscle myosin II

1995 ◽  
Vol 16 (5) ◽  
pp. 491-498 ◽  
Author(s):  
Kevin A. Edwards ◽  
Xiao-Jia Chang ◽  
Daniel P. Kiehart
Keyword(s):  
Light Chain ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Essential Light Chain ◽  
Nonmuscle Myosin Ii

scholarly journals Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

1997 ◽  
Vol 139 (7) ◽  
pp. 1805-1819 ◽  
Author(s):  
Pascale Jordan ◽  
Roger Karess
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Germ Line ◽  
Myosin Ii ◽  
Oocyte Development ◽  
Nurse Cells ◽  
Ring Canals

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.

scholarly journals Binding of a Newly Identified Essential Light Chain to Expressed Plasmodium falciparum Class XIV Myosin Enhances Actin Motility

2017 ◽  
Author(s):  
Carol S. Bookwalter ◽  
Chwen L. Tay ◽  
Rama McCrorie ◽  
Michael J. Previs ◽  
Elena B. Krementsova ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Causative Agent ◽  
Heavy Chain ◽  
Light Chain ◽  
Expression System ◽  
Apicomplexan Parasite ◽  
Host Cells ◽  
Duty Ratio ◽  
Essential Light Chain ◽  
Maximal Speed

AbstractMotility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional “essential” light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 μm/sec, more than twice that of PfMyoA-MTIP (~1.7 μm/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.SignificanceMotility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, relies on a divergent actomyosin system powered by the class XIV myosin, PfMyoA. We show that functional PfMyoA can be expressed in Sf9 cells if a Plasmodium spp. myosin chaperone is co-expressed. We identified an “essential” light chain (PfELC) that binds to PfMyoA in parasites. In vitro expression of PfMyoA heavy chain with PfELC and the known light chain MTIP produced the fastest speeds of actin movement (~3.8 μm/sec). Duty ratio estimates suggest that ~6 PfMyoA motors can move actin at maximal speed, a feature that may facilitate interaction with short, dynamic Plasmodium actin filaments. Our findings enable drug screening for myosin-based inhibitors of Plasmodium cellular invasion.

scholarly journals A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila

2020 ◽  
Vol 133 (21) ◽  
pp. jcs252965
Author(s):  
Stefano Sechi ◽  
Anna Frappaolo ◽  
Angela Karimpour-Ghahnavieh ◽  
Roberta Fraschini ◽  
Maria Grazia Giansanti
Keyword(s):  
Plasma Membrane ◽  
Heavy Chain ◽  
Light Chain ◽  
Animal Cell ◽  
Myosin Ii ◽  
Contractile Ring ◽  
Ring Assembly ◽  
Missense Allele ◽  
Phosphatidylinositol 4 ◽  
Muscle Myosin

ABSTRACTIn animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P–GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

scholarly journals Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian θ isoforms

2002 ◽  
Vol 368 (3) ◽  
pp. 809-815 ◽  
Author(s):  
Marc A. de la ROCHE ◽  
Janet L. SMITH ◽  
Maribel RICO ◽  
Silvia CARRASCO ◽  
Isabel MERIDA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Catalytic Domain ◽  
Single Gene ◽  
Myosin Ii ◽  
Terminal Segment ◽  
Gene Encoding ◽  
Kinase Gene ◽  
Myosin Heavy Chain Protein

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the θ subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.

scholarly journals The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

1998 ◽  
Vol 143 (5) ◽  
pp. 1239-1247 ◽  
Author(s):  
Susan M. Beckwith ◽  
Christian H. Roghi ◽  
Bo Liu ◽  
N. Ronald Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Light Chain ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dynein Light Chain ◽  
Myosin V ◽  
Dynein Heavy Chain

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.

scholarly journals Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis

2012 ◽  
Vol 417 (2) ◽  
pp. 686-691 ◽  
Author(s):  
Tomo Kondo ◽  
Rieko Isoda ◽  
Takashi Uchimura ◽  
Mutsumi Sugiyama ◽  
Kozue Hamao ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Myosin Ii ◽  
Regulatory Light Chain

Structural abnormalities develop in the brain after ablation of the gene encoding nonmuscle myosin II-B heavy chain

2001 ◽  
Vol 433 (1) ◽  
pp. 62-74 ◽  
Author(s):  
Antonella N. Tullio ◽  
Paul C. Bridgman ◽  
Nancy J. Tresser ◽  
Chi-Chao Chan ◽  
Mary Anne Conti ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Gene Encoding ◽  
Nonmuscle Myosin Ii ◽  
Structural Abnormalities ◽  
The Brain

scholarly journals A variant chromosome translocation at 11q13 identifying PRAD1/cyclin D1 as the BCL-1 gene

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1226-1231 ◽  
Author(s):  
H Komatsu ◽  
S Iida ◽  
K Yamamoto ◽  
C Mikuni ◽  
M Nitta ◽  
...  
Keyword(s):  
Cyclin D1 ◽  
Heavy Chain ◽  
Light Chain ◽  
Chromosome Translocation ◽  
Transcriptional Unit ◽  
Chain Gene ◽  
Gene Encoding ◽  
Light Chain Gene ◽  
Variant Chromosome ◽  
Ig Heavy Chain

Abstract The 11q13 breakpoint region of t(11;14) (q13;q32), translocated to the Ig heavy chain locus at 14q32, has been designated as BCL-1 for B-cell leukemia/lymphoma-1, but the nature of the transcriptional unit has long remained unclear. Recently, the PRAD1 gene encoding cyclin D1, isolated from the 11q13 region, was proposed as a candidate BCL-1 gene on the basis of chromosome walking and concordant overexpression of PRAD1 mRNA in cell lines with t(11;14)(q13;q32). We report here molecular analysis of a variant translocation at the BCL-1 locus, t(11;22)(q13;q11), showing juxtaposition of the Ig light chain gene, Ig lambda, to the PRAD1 gene at its 3′ end, resulting in overexpression of PRAD1 mRNA. Because only the PRAD1 gene is present between the Ig heavy chain and light chain gene breakpoints, an identity between BCL-1 and the PRAD1/cyclin D1 gene is strongly indicated.

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