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1.
Nat Commun ; 15(1): 2755, 2024 Mar 29.
Article in English | MEDLINE | ID: mdl-38553438

ABSTRACT

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence. Here we demonstrate the power of props by functional imaging within distinct regions of the zebrafish brain, monitoring calcium firing inside muscle cells of moving Drosophila larvae, super-resolution imaging of selected cell layers, and by optically unwrapping the curved surface of a Drosophila embryo. We anticipate that props will accelerate volumetric interrogation, ranging from subcellular to mesoscopic scales.


Subject(s)
Drosophila , Zebrafish , Animals , Microscopy, Fluorescence/methods , Brain/ultrastructure , Larva
2.
Dev Cell ; 58(19): 1864-1879.e4, 2023 10 09.
Article in English | MEDLINE | ID: mdl-37729921

ABSTRACT

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. Here, we demonstrate that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. We show that while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis.


Subject(s)
Drosophila Proteins , Hippo Signaling Pathway , Animals , Actomyosin/metabolism , Cell Polarity , Drosophila/metabolism , Drosophila Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction
3.
Elife ; 112022 12 19.
Article in English | MEDLINE | ID: mdl-36533896

ABSTRACT

In the early Caenorhabditis elegans embryo, cell polarization and cytokinesis are interrelated yet distinct processes. Here, we sought to understand a poorly understood aspect of cleavage furrow positioning. Early C. elegans embryos deficient in the cytokinetic regulator centralspindlin form furrows, due to an inhibitory activity that depends on aster positioning relative to the polar cortices. Here, we show polar relaxation is associated with depletion of cortical ECT-2, a RhoGEF, specifically at the posterior cortex. Asymmetric ECT-2 accumulation requires intact centrosomes, Aurora A (AIR-1), and myosin-dependent cortical flows. Within a localization competent ECT-2 fragment, we identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. During both polarization and cytokinesis, our results suggest that centrosomal AIR-1 breaks symmetry via ECT-2 phosphorylation; this local inhibition of ECT-2 is amplified by myosin-driven flows that generate regional ECT-2 asymmetry. Together, these mechanisms cooperate to induce polarized assembly of cortical myosin, contributing to both embryo polarization and cytokinesis.


Subject(s)
Caenorhabditis elegans Proteins , Cytokinesis , Animals , Cytokinesis/physiology , Caenorhabditis elegans/physiology , Myosins , Rho Guanine Nucleotide Exchange Factors , Guanine Nucleotide Exchange Factors , Aurora Kinase A
4.
Small GTPases ; 12(5-6): 416-428, 2021.
Article in English | MEDLINE | ID: mdl-33985411

ABSTRACT

Epithelial folding is a common means to execute morphogenetic movements. The gastrulating Drosophila embryo offers many examples of epithelial folding events, including the ventral, cephalic, and dorsal furrows. Each of these folding events is associated with changes in intracellular contractility and/or cytoskeleton structures that autonomously promote epithelial folding. Here, we review accumulating evidence that suggests the progression and final form of ventral, cephalic, and dorsal furrows are also influenced by the behaviour of cells neighbouring these folds. We further discuss the prevalence and importance of junctional rearrangements during epithelial folding events, suggesting adherens junction components are prime candidates to modulate the transmission of the intercellular forces that influence folding events. Finally, we discuss how recently developed methods that enable precise spatial and/or temporal control of protein activity allow direct testing of molecular models of morphogenesis in vivo.


Subject(s)
Cytoskeleton/physiology , Drosophila Proteins/metabolism , Drosophila/physiology , Embryo, Nonmammalian/physiology , Epithelial Cells/physiology , Monomeric GTP-Binding Proteins/metabolism , Morphogenesis , Animals , Cytoskeleton/enzymology , Drosophila/enzymology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/enzymology , Epithelial Cells/enzymology , Microtubules/enzymology , Microtubules/physiology
5.
J Cell Sci ; 134(3)2021 02 08.
Article in English | MEDLINE | ID: mdl-33468621

ABSTRACT

Haploid male gametes are produced through meiosis during gametogenesis. Whereas the cell biology of mitosis and meiosis is well studied in the nematode Caenorhabditis elegans, comparatively little is known regarding the physical division of primary spermatocytes during meiosis I. Here, we investigated this process using high-resolution time-lapse confocal microscopy and examined the spatiotemporal regulation of contractile ring assembly in C. elegans primary spermatocytes. We found that centralspindlin and RhoA effectors were recruited to the equatorial cortex of dividing primary spermatocytes for contractile ring assembly before segregation of homologous chromosomes. We also observed that perturbations shown to promote centralspindlin oligomerization regulated the cortical recruitment of NMY-2 and impacted the order in which primary spermatocytes along the proximal-distal axis of the gonad enter meiosis I. These results expand our understanding of the cellular division of primary spermatocytes into secondary spermatocytes during meiosis I.This article has an associated First Person interview with the first author of the paper.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cytokinesis , Male , Meiosis , Spermatocytes
6.
Elife ; 92020 11 17.
Article in English | MEDLINE | ID: mdl-33200987

ABSTRACT

Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Gastrulation/physiology , rho GTP-Binding Proteins/metabolism , Animals , Cell Line , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Embryo, Nonmammalian , Epithelium , Gene Expression Regulation, Developmental , Larva , Organisms, Genetically Modified , rho GTP-Binding Proteins/genetics
7.
Elife ; 92020 02 14.
Article in English | MEDLINE | ID: mdl-32057294

ABSTRACT

Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. We demonstrate that posterior localization of osk/Staufen is determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk/Staufen from the cortex along microtubules, myosin-V anchors osk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density.


One of the most fundamental steps of embryonic development is deciding which end of the body should be the head, and which should be the tail. Known as 'axis specification', this process depends on the location of genetic material called mRNAs. In fruit flies, for example, the tail-end of the embryo accumulates an mRNA called oskar. If this mRNA is missing, the embryo will not develop an abdomen. The build-up of oskar mRNA happens before the egg is even fertilized and depends on two types of scaffold proteins in the egg cell called microtubules and microfilaments. These scaffolds act like 'train tracks' in the cell and have associated protein motors, which work a bit like trains, carrying cargo as they travel up and down along the scaffolds. For microtubules, one of the motors is a protein called kinesin-1, whereas for microfilaments, the motors are called myosins. Most microtubules in the egg cell are pointing away from the membrane, while microfilament tracks form a dense network of randomly oriented filaments just underneath the membrane. It was already known that kinesin-1 and a myosin called myosin-V are important for localizing oskar mRNA to the posterior of the egg. However, it was not clear why the mRNA only builds up in that area. To find out, Lu et al. used a probe to track oskar mRNA, while genetically manipulating each of the motors so that their ability to transport cargo changed. Modulating the balance of activity between the two motors revealed that kinesin-1 and myosin-V engage in a tug-of-war inside the egg: myosin-V tries to keep oskar mRNA underneath the membrane of the cell, while kinesin-1 tries to pull it away from the membrane along microtubules. The winner of this molecular battle depends on the number of microtubule tracks available in the local area of the cell. In most parts of the cell, there are abundant microtubules, so kinesin-1 wins and pulls oskar mRNA away from the membrane. But at the posterior end of the cell there are fewer microtubules, so myosin-V wins, allowing oskar mRNA to localize in this area. Artificially 'shaving' some microtubules in a local area immediately changed the outcome of this tug-of-war creating a build-up of oskar mRNA in the 'shaved' patch. This is the first time a molecular tug-of-war has been shown in an egg cell, but in other types of cell, such as neurons and pigment cells, myosins compete with kinesins to position other molecular cargoes. Understanding these processes more clearly sheds light not only on embryo development, but also on cell biology in general.


Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/genetics , Kinesins/physiology , Myosin Type V/physiology , Animals , Drosophila Proteins/metabolism , Female , Kinesins/metabolism , Male , Microscopy, Electron , Microtubules/metabolism , Microtubules/physiology , Myosin Type V/metabolism , Oocytes/metabolism , Oocytes/physiology , Optogenetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiology
8.
J Cell Biol ; 218(4): 1250-1264, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30728176

ABSTRACT

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.


Subject(s)
Cell Cycle Proteins/metabolism , Cytokinesis , Microtubule-Associated Proteins/metabolism , Microtubules/enzymology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/enzymology , Animals , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Cycle Proteins/genetics , Enzyme Activation , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Phosphoproteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Transport , Proto-Oncogene Proteins/genetics , Signal Transduction , Spindle Apparatus/genetics , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , Polo-Like Kinase 1
9.
Curr Biol ; 28(9): R570-R580, 2018 05 07.
Article in English | MEDLINE | ID: mdl-29738735

ABSTRACT

The active form of the small GTPase RhoA is necessary and sufficient for formation of a cytokinetic furrow in animal cells. Despite the conceptual simplicity of the process, the molecular mechanisms that control it are intricate and involve redundancy at multiple levels. Here, we discuss our current knowledge of the mechanisms underlying spatiotemporal regulation of RhoA during cytokinesis by upstream activators. The direct upstream activator, the RhoGEF Ect2, requires activation due to autoinhibition. Ect2 is primarily activated by the centralspindlin complex, which contains numerous domains that regulate its subcellular localization, oligomeric state, and Ect2 activation. We review the functions of these domains and how centralspindlin is regulated to ensure correctly timed, equatorial RhoA activation. Highlighting recent evidence, we propose that although centralspindlin does not always prominently accumulate on the plasma membrane, it is the site where it promotes RhoA activation during cytokinesis.


Subject(s)
Cytokinesis/physiology , rhoA GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/physiology , Animals , Cell Membrane/metabolism , Embryonic Development/physiology , GTP Phosphohydrolases/metabolism , Humans , Microtubule-Associated Proteins/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , Spatio-Temporal Analysis , Spindle Apparatus/metabolism
10.
F1000Res ; 6: 1788, 2017.
Article in English | MEDLINE | ID: mdl-29043078

ABSTRACT

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early Caenorhabditis elegans embryo, RhoA activation and furrow ingression require the centralspindlin complex. This exception is due to the existence of a parallel pathway for RhoA activation in C. elegans. Centralspindlin contains CYK-4 which contains a predicted Rho family GTPase-activating protein (GAP) domain. The function of this domain has been the subject of considerable debate. Some publications suggest that the GAP domain promotes RhoA activation (for example, Zhang and Glotzer, 2015; Loria, Longhini and Glotzer, 2012), whereas others suggest that it functions to inactivate the GTPase Rac1 (for example, Zhuravlev et al., 2017). Here, we review the mechanisms underlying RhoA activation during cytokinesis, primarily focusing on data in C. elegans. We highlight the importance of considering the parallel pathway for RhoA activation and detailed analyses of  cyk-4 mutant phenotypes when evaluating the role of the GAP domain of CYK-4.

11.
Elife ; 62017 07 06.
Article in English | MEDLINE | ID: mdl-28682236

ABSTRACT

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle , Guanine Nucleotide Exchange Factors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/metabolism , Cell Polarity , Optogenetics , Protein Multimerization
12.
Nat Commun ; 8: 15817, 2017 06 12.
Article in English | MEDLINE | ID: mdl-28604737

ABSTRACT

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.


Subject(s)
Stress Fibers/physiology , Zyxin/physiology , rhoA GTP-Binding Protein/physiology , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/physiology , Animals , Mechanotransduction, Cellular , Mice , NIH 3T3 Cells , Optogenetics , Stress Fibers/metabolism , Zyxin/genetics , Zyxin/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
13.
Article in English | MEDLINE | ID: mdl-28007751

ABSTRACT

SUMMARYCell division-cytokinesis-involves large-scale rearrangements of the entire cell. Primarily driven by cytoskeletal proteins, cytokinesis also depends on topological rearrangements of the plasma membrane, which are coordinated with nuclear division in both space and time. Despite the fundamental nature of the process, different types of eukaryotic cells show variations in both the structural mechanisms of cytokinesis and the regulatory controls. In animal cells and fungi, a contractile actomyosin-based structure plays a central, albeit flexible, role. Here, the underlying molecular mechanisms are summarized and integrated and common themes are highlighted.


Subject(s)
Cytokinesis , Fungi/metabolism , Animals
15.
J Cell Biol ; 213(6): 641-9, 2016 06 20.
Article in English | MEDLINE | ID: mdl-27298323

ABSTRACT

The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. Although many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. We have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation. Light-mediated recruitment of a RhoGEF domain to the plasma membrane leads to rapid induction of RhoA activity, leading to assembly of cytokinetic furrows that partially ingress. Furthermore, furrow formation in response to RhoA activation is not temporally or spatially restricted. RhoA activation is sufficient to generate furrows at both the cell equator and cell poles, in both metaphase and anaphase. Remarkably, furrow formation can be initiated in rounded interphase cells, but not adherent cells. These results indicate that RhoA activation is sufficient to induce assembly of functional contractile rings and that cell rounding facilitates furrow formation.


Subject(s)
Anaphase/physiology , Cytokinesis/physiology , Interphase/physiology , Metaphase/physiology , Spindle Apparatus/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , HeLa Cells , Humans , Mice , Microtubule-Associated Proteins/metabolism , NIH 3T3 Cells , Spindle Apparatus/physiology
16.
ACS Synth Biol ; 5(7): 554-60, 2016 07 15.
Article in English | MEDLINE | ID: mdl-26513473

ABSTRACT

The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.


Subject(s)
Avena/chemistry , Peroxisomes/metabolism , Phototropins/metabolism , Protein Transport , Recombinant Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Light , Molecular Imaging/methods , Phototropins/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Domains , Protein Engineering/methods , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Proteins/genetics , Two-Hybrid System Techniques
17.
Curr Biol ; 25(24): R1183-5, 2015 Dec 21.
Article in English | MEDLINE | ID: mdl-26702657

ABSTRACT

A Caenorhabditis elegans mutant has been identified in which an ectopic myosin cap shifts the cleavage furrow relative to the spindle center. Surprisingly, the molecules that suppress this cap in wild-type embryos generate a cap in other asymmetrically dividing cells.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Cytokinesis/physiology , Myosins/metabolism , Spindle Apparatus/metabolism , Animals
18.
Elife ; 42015 Aug 07.
Article in English | MEDLINE | ID: mdl-26252513

ABSTRACT

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , Cytokinesis , rhoA GTP-Binding Protein/metabolism , Animals , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors
19.
Dev Cell ; 33(2): 204-15, 2015 Apr 20.
Article in English | MEDLINE | ID: mdl-25898168

ABSTRACT

In metazoans, cytokinesis is triggered by activation of the GTPase RhoA at the equatorial plasma membrane. ECT-2, the guanine nucleotide exchange factor (GEF) required for RhoA activation, is activated by the centralspindlin complex that concentrates on spindle midzone microtubules. However, these microtubules and the plasma membrane are not generally in apposition, and thus the mechanism by which RhoA is activated at the cell equator remains unknown. Here we report that a regulated pool of membrane-bound, oligomeric centralspindlin stimulates RhoA activation. The membrane-binding C1 domain of CYK-4, a centralspindlin component, promotes furrow initiation in C. elegans embryos and human cells. Membrane localization of centralspindlin oligomers is globally inhibited by PAR-5/14-3-3. This activity is antagonized by the chromosome passenger complex (CPC), resulting in RhoA activation at the nascent cleavage site. Therefore, CPC-directed centralspindlin oligomerization during anaphase induces contractile ring assembly at the membrane.


Subject(s)
Aurora Kinase B/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Cytokinesis/genetics , rhoA GTP-Binding Protein/metabolism , Animals , Aurora Kinase B/genetics , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , Cell Line, Tumor , Cell Membrane/metabolism , Guanine Nucleotide Exchange Factors/metabolism , HeLa Cells , Humans , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Protein Multimerization , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering , Spindle Apparatus/genetics
20.
J Biol Chem ; 288(27): 19785-95, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23720745

ABSTRACT

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.


Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/chemistry , Kinesins/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Spindle Apparatus/chemistry , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Electron Spin Resonance Spectroscopy , Kinesins/genetics , Kinesins/metabolism , Microtubules/genetics , Microtubules/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Binding , Protein Structure, Quaternary , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
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