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1.
PLoS Genet ; 19(3): e1010319, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36976799

RESUMO

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and ß-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved.


Assuntos
Actomiosina , Caenorhabditis elegans , Animais , Actomiosina/genética , Actomiosina/metabolismo , Zixina/genética , Zixina/metabolismo , Caenorhabditis elegans/metabolismo , Constrição , Proteômica , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Morfogênese/genética
2.
Development ; 140(23): 4672-82, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24173802

RESUMO

Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.


Assuntos
Córtex Cerebral/metabolismo , Citocinese/fisiologia , Cinesinas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Polaridade Celular/genética , Expressão Gênica , Cinesinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , RNA Mensageiro/biossíntese
3.
Neural Dev ; 12(1): 5, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28359322

RESUMO

BACKGROUND: How neurons change their cytoskeleton to adopt their complex polarized morphology is still not understood. Growing evidence suggests that proteins that help build microtubule structures during cell division are also involved in building and remodeling the complex cytoskeletons of neurons. Kif20b (previously called MPP1 or Mphosph1) is the most divergent member of the Kinesin-6 family of "mitotic" kinesins that also includes Kif23/MKLP1 and Kif20a/MKLP2. We previously isolated a loss-of-function mouse mutant of Kif20b and showed that it had a thalamocortical axon guidance defect and microcephaly. METHODS: We demonstrate here, using the mouse mutant, that Kif20b is required for neuron morphogenesis in the embryonic neocortex. In vivo and in vitro cortical neurons were labeled and imaged to analyze various aspects of morphogenesis. RESULTS: Loss of Kif20b disrupts polarization as well as neurite outgrowth, branching and caliber. In vivo, mutant cortical neurons show defects in orientation, and have shorter thinner apical dendrites that branch closer to the cell body. In vitro, without external polarity cues, Kif20b mutant neurons show a strong polarization defect. This may be due in part to loss of the polarity protein Shootin1 from the axonal growth cone. Those mutant neurons that do succeed in polarizing have shorter axons with more branches, and longer minor neurites. These changes in shape are not due to alterations in cell fate or neuron layer type. Surprisingly, both axons and minor neurites of mutant neurons have increased widths and longer growth cone filopodia, which correlate with abnormal microtubule organization. Live analysis of axon extension shows that Kif20b mutant axons display more variable growth with increased retraction. CONCLUSIONS: These results demonstrate that Kif20b is required cell-autonomously for proper morphogenesis of cortical pyramidal neurons. Kif20b regulates neuron polarization, and axon and dendrite branching, outgrowth, and caliber. Kif20b protein may act by bundling microtubules into tight arrays and by localizing effectors such as Shootin1. Thus it may help shape neurites, sustain consistent axon growth, and inhibit branching. This work advances our understanding of how neurons regulate their cytoskeleton to build their elaborate shapes. Finally, it suggests that neuronal connectivity defects may be present in some types of microcephaly.


Assuntos
Polaridade Celular , Córtex Cerebral/embriologia , Cinesinas/genética , Morfogênese , Células Piramidais/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Cones de Crescimento/metabolismo , Camundongos , Microtúbulos/fisiologia , Mutação , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Pseudópodes/fisiologia , Células Piramidais/citologia
4.
Curr Biol ; 26(16): 2079-89, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27451898

RESUMO

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, ß-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação ao GTP/genética , Gastrulação , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Actomiosina/metabolismo , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proteínas de Ligação ao GTP/metabolismo , Junções Intercelulares/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
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