RESUMO
Molecular motors drive cytoskeletal rearrangements to change cell shape. Myosins are the motors that move, cross-link, and modify the actin cytoskeleton. The primary force generator in contractile actomyosin networks is nonmuscle myosin II (NMMII), a molecular motor that assembles into ensembles that bind, slide, and cross-link actin filaments (F-actin). The multivalence of NMMII ensembles and their multiple roles have confounded the resolution of crucial questions, including how the number of NMMII subunits affects dynamics and what affects the relative contribution of ensembles' cross-linking versus motoring activities. Because biophysical measurements of ensembles are sparse, modeling of actomyosin networks has aided in discovering the complex behaviors of NMMII ensembles. Myosin ensembles have been modeled via several strategies with variable discretization or coarse graining and unbinding dynamics, and although general assumptions that simplify motor ensembles result in global contractile behaviors, it remains unclear which strategies most accurately depict cellular activity. Here, we used an agent-based platform, Cytosim, to implement several models of NMMII ensembles. Comparing the effects of bond type, we found that ensembles of catch-slip and catch motors were the best force generators and binders of filaments. Slip motor ensembles were capable of generating force but unbound frequently, resulting in slower contractile rates of contractile networks. Coarse graining of these ensemble types from two sets of 16 motors on opposite ends of a stiff rod to two binders, each representing 16 motors, reduced force generation, contractility, and the total connectivity of filament networks for all ensemble types. A parallel cluster model, previously used to describe ensemble dynamics via statistical mechanics, allowed better contractility with coarse graining, though connectivity was still markedly reduced for this ensemble type with coarse graining. Together, our results reveal substantial tradeoffs associated with the process of coarse graining NMMII ensembles and highlight the robustness of discretized catch-slip ensembles in modeling actomyosin networks.
Assuntos
Actomiosina , Miosina Tipo II , Citoesqueleto de Actina , Actinas , Contração Muscular , MiosinasRESUMO
Epithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.
Assuntos
Junções Aderentes/metabolismo , Citoesqueleto/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Células CACO-2 , Proteínas de Ligação a DNA , Cães , Forminas/metabolismo , Humanos , Células Madin Darby de Rim Canino , Modelos Biológicos , Quinase de Cadeia Leve de Miosina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Divisão Celular/fisiologia , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Anáfase/fisiologia , Animais , Aurora Quinase B , Caenorhabditis elegans/citologia , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA/fisiologia , Fuso Acromático/química , Fuso Acromático/metabolismoRESUMO
Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume.
Assuntos
Caenorhabditis elegans/embriologia , Tamanho Celular , Embrião não Mamífero/fisiologia , Microtúbulos/fisiologia , Paracentrotus/embriologia , Fuso Acromático/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Embrião não Mamífero/citologia , Paracentrotus/fisiologiaRESUMO
Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.
Assuntos
Luz , Microscopia de Fluorescência/métodos , Fotodegradação , Animais , Arabidopsis/citologia , Linhagem Celular , Núcleo Celular/metabolismo , Fluorescência , Fungos/citologia , Humanos , Imageamento Tridimensional , Modelos Biológicos , Reprodutibilidade dos Testes , Imagem com Lapso de TempoRESUMO
To study the dynamics of stress fiber components in cultured fibroblasts, we expressed alpha-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and alpha-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and alpha-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.
Assuntos
Actinina/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estresse/fisiologia , Fibras de Estresse/ultraestrutura , Actinina/análise , Actinina/genética , Animais , Fibroblastos/imunologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Lisofosfolipídeos/farmacologia , Toxinas Marinhas , Camundongos , Cadeias Leves de Miosina/análise , Cadeias Leves de Miosina/genética , Miosina Tipo II/análise , Miosina Tipo II/genética , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Fotodegradação , Células Swiss 3T3RESUMO
Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain-containing protein ZYG-9(TOGp) is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.
Assuntos
Microtúbulos/metabolismo , Microtúbulos/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Ciclinas/metabolismo , Desaceleração , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Modelos Moleculares , Polimerização , Estatmina , Tubulina (Proteína)/metabolismoRESUMO
Microtubules (MTs) are cytoskeletal filaments essential for many processes in eukaryotic cells. Assembled of tubulin subunits, MTs are dynamic structures that undergo successive and stochastic phases of polymerization and depolymerization, a behavior called dynamic instability. Dynamic instability has been extensively studied in cultured cells and in vitro using cytoplasmic extracts or reconstituted MTs. However, how MTs behave in intact tissues and how their dynamics are affected by or affect tissue function are poorly understood. Recent advances in high-resolution live imaging have helped overcome technical limitation in order to visualize MTs in intact living organisms including Drosophila or Caenorhabditis elegans. We recently took advantage of the well-characterized development, small size and transparency of C. elegans to monitor MT dynamics throughout tissue biogenesis with high spatial and temporal resolution. Using the sex myoblast lineage that generates the egg-laying muscles from 2 mitotic precursors, we identified selective dynamics in precursor versus differentiated cells, and molecular regulation of MT dynamics changes that occur during cell differentiation. We discuss here how this approach led to novel insights into the regulation of MTs dynamics and organization in vivo.
RESUMO
The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Citocinese/fisiologia , Animais , Caenorhabditis elegans/citologia , Drosophila melanogaster , Feminino , Células HeLa , HumanosRESUMO
Microtubules (MTs) are cytoskeletal polymers that undergo dynamic instability, the stochastic transition between growth and shrinkage phases. MT dynamics are required for diverse cellular processes and, while intrinsic to tubulin, are highly regulated. However, little is known about how MT dynamics facilitate or are regulated by tissue biogenesis and differentiation. We imaged MT dynamics in a smooth muscle-like lineage in intact developing Caenorhabditis elegans. All aspects of MT dynamics change significantly as stem-like precursors exit mitosis and, secondarily, as they differentiate. We found that suppression, but not enhancement, of dynamics perturbs differentiated muscle function in vivo. Distinct ensembles of MT-associated proteins are specifically required for tissue biogenesis versus tissue function. A CLASP family MT stabilizer and the depolymerizing kinesin MCAK are differentially required for MT dynamics in the precursor or differentiated cells, respectively. All of these multidimensional phenotypic comparisons were facilitated by a data display method called the diamond graph.
Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Animais , Proteínas de Caenorhabditis elegans/fisiologia , Linhagem da Célula/fisiologia , Cinesinas/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Óvulo/fisiologia , Reprodução/fisiologia , Fuso Acromático/fisiologia , Tubulina (Proteína)/fisiologiaRESUMO
Cytokinesis generally produces two separate daughter cells, but in some tissues daughter nuclei remain connected to a shared cytoplasm, or syncytium, through incomplete cytokinesis. How syncytia form remains poorly understood. We studied syncytial formation in the Caenorhabditis elegans germline, in which germ cells connect to a shared cytoplasm core (the rachis) via intercellular bridges. We found that syncytial architecture initiates early in larval development, and germ cells become progressively interconnected until adulthood. The short Anillin family scaffold protein ANI-2 is enriched at intercellular bridges from the onset of germ cell specification, and ANI-2 loss resulted in destabilization of intercellular bridges and germ cell multinucleation defects. These defects were partially rescued by depleting the canonical Anillin ANI-1 or blocking cytoplasmic streaming. ANI-2 is also required for elastic deformation of the gonad during ovulation. We propose that ANI-2 promotes germ cell syncytial organization and allows for compensation of the mechanical stress associated with oogenesis by conferring stability and elasticity to germ cell intercellular bridges.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/metabolismo , Células Gigantes/fisiologia , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/fisiologia , Animais , Caenorhabditis elegans/citologia , Corrente Citoplasmática , Feminino , Células Germinativas/metabolismo , Gônadas/citologia , Larva/citologia , Larva/metabolismo , Masculino , Oogênese , Estabilidade ProteicaRESUMO
Differential interference contrast (DIC) imaging of Caenorhabditis elegans embryogenesis led to a Nobel Prize in Physiology or Medicine (Sulston et al., 1983) as did the first use of green fluorescent protein (GFP) in a transgenic C. elegans (Chalfie et al., 1994). Given that C. elegans is free living, does not require exceptional environmental control, and is optically clear, live imaging is a powerful tool in for this model system. Combining genetics with high-resolution imaging has continued to make important contributions to many fields. In this chapter, we discuss how certain aspects of high-resolution microscopy are implemented. This is not an exhaustive review of microscopy; it is meant to be a helpful guide and point of reference for some basic concepts in imaging. While these concepts are largely true for all biological imaging, they are chosen as particularly important for C. elegans.
Assuntos
Caenorhabditis elegans/ultraestrutura , Desenvolvimento Embrionário/fisiologia , Larva/ultraestrutura , Imagem Molecular/métodos , Animais , Caenorhabditis elegans/embriologia , Sistemas Computacionais , Embrião não Mamífero , Fixadores , Proteínas de Fluorescência Verde , Larva/fisiologia , Microscopia , Microscopia Confocal , Modelos Biológicos , Imagem Molecular/instrumentaçãoRESUMO
The mitotic spindle, due to its striking form, has been imaged for well over 100 years. Composed largely of microtubules and chromosomes, the spindle also contains numerous proteins whose roles include biochemical and biophysical regulation of mitosis. Given the transient, dynamic nature of the spindle, the light microscope continues to be the main tool employed to unlock its mysteries. In this chapter, we will discuss modern light microscopy techniques commonly used for imaging this intricate cellular machine as well as provide examples and protocols. We will also describe some biological preparations and experimental regimes for investigation of the mitotic spindle.
Assuntos
Rastreamento de Células/métodos , Fuso Acromático/metabolismo , Animais , Caenorhabditis elegans/citologia , Divisão Celular/genética , Linhagem Celular , Recuperação de Fluorescência Após Fotodegradação , Humanos , Mamíferos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microscopia de Contraste de Fase/métodos , Microscopia de Polarização/métodos , Microscopia de Vídeo/métodos , Microtúbulos/genética , Mitose/genética , Saccharomyces cerevisiae/citologia , Fuso Acromático/genéticaRESUMO
The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora B(AIR-2) and the centralspindlin component MKLP1(ZEN-4) causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora B(AIR-2)-inhibited embryos, whereas inhibition of Rac does so in MKLP1(ZEN-4)-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Citocinese , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Fuso Acromático/metabolismo , Animais , Aurora Quinase B , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/ultraestrutura , Genótipo , Cinesinas/antagonistas & inibidores , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: The serine/threonine kinase LKB1 regulates cell growth and polarity in metazoans, and loss of LKB1 function is implicated in the development of some epithelial cancers. Despite its fundamental role, the mechanism by which LKB1 regulates polarity establishment and/or maintenance is unclear. In the present study, we use the nematode C. elegans to investigate the role of the LKB1 ortholog PAR-4 in actomyosin contractility, a cellular process essential for polarity establishment and cell division in the early embryo. RESULTS: Using high-resolution time-lapse imaging of GFP-tagged nonmuscle myosin II (NMY-2), we found that par-4 mutations reduce actomyosin contractility during polarity establishment, leading to the mispositioning of anterior PAR proteins and to defects in contractile ring ingression during cytokinesis. Fluorescence recovery after photobleaching analysis revealed that the mobility of a cortical population of NMY-2 was reduced in par-4 mutants. Interestingly, the contractility defects of par-4 mutants depend on the reciprocal activity of ANI-1 and ANI-2, two C. elegans homologs of the actin cytoskeletal scaffold protein anillin. CONCLUSION: Because loss of PAR-4 promoted inappropriate accumulation of ANI-2 at the cell cortex, we propose that PAR-4 controls C. elegans embryonic polarity by regulating the activity of anillin family scaffold proteins, thus enabling turnover of cortical myosin and efficient actomyosin contractility. This work provides the first description of a cellular mechanism by which PAR-4/LKB1 mediates cell polarization.
Assuntos
Actomiosina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Citocinese/fisiologia , Proteínas dos Microfilamentos/metabolismo , Actomiosina/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Polaridade Celular , Proteínas dos Microfilamentos/genética , Mutação , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas Serina-Treonina QuinasesRESUMO
Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. Here, we show that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, we found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A.