RESUMEN
Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditiselegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)-dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.
Asunto(s)
Polaridad Celular , Células Epiteliales , Células Epiteliales/metabolismo , Uniones Intercelulares , Mucosa Intestinal , Intestinos , EpitelioRESUMEN
E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix-loop-helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure-function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Procesamiento Proteico-Postraduccional , Proteolisis , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Dimerización , Regulación hacia Abajo , Femenino , Humanos , Estabilidad Proteica , Útero/embriologíaRESUMEN
Non-centrosomal microtubule organizing centers (ncMTOCs) are found in most differentiated cells, but how these structures regulate microtubule organization and dynamics is largely unknown. We optimized a tissue-specific degradation system to test the role of the essential centrosomal microtubule nucleators γ-tubulin ring complex (γ-TuRC) and AIR-1/Aurora A at the apical ncMTOC, where they both localize in Caenorhabditis elegans embryonic intestinal epithelial cells. As at the centrosome, the core γ-TuRC component GIP-1/GCP3 is required to recruit other γ-TuRC components to the apical ncMTOC, including MZT-1/MZT1, characterized here for the first time in animal development. In contrast, AIR-1 and MZT-1 were specifically required to recruit γ-TuRC to the centrosome, but not to centrioles or to the apical ncMTOC. Surprisingly, microtubules remain robustly organized at the apical ncMTOC upon γ-TuRC and AIR-1 co-depletion, and upon depletion of other known microtubule regulators, including TPXL-1/TPX2, ZYG-9/ch-TOG, PTRN-1/CAMSAP, and NOCA-1/Ninein. However, loss of GIP-1 removed a subset of dynamic EBP-2/EB1-marked microtubules, and the remaining dynamic microtubules grew faster. Together, these results suggest that different microtubule organizing centers (MTOCs) use discrete proteins for their function, and that the apical ncMTOC is composed of distinct populations of γ-TuRC-dependent and -independent microtubules that compete for a limited pool of resources.
Asunto(s)
Centrosoma/metabolismo , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/metabolismo , Animales , Aurora Quinasa A , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Centrosoma/fisiología , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Proteínas Asociadas a Microtúbulos , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Tubulina (Proteína)/metabolismoRESUMEN
Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.
Asunto(s)
Axonema , Caenorhabditis elegans , Animales , Ratones , Axonema/metabolismo , Axonema/ultraestructura , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Mamíferos , Microtúbulos/metabolismo , Movimiento , Tubulina (Proteína)/metabolismoRESUMEN
Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.
RESUMEN
Many ant species are equipped with chemical defenses, although how these compounds impact nervous system function is unclear. Here, we examined the utility of Caenorhabditis elegans chemotaxis assays for investigating how ant chemical defense compounds are detected by heterospecific nervous systems. We found that C. elegans respond to extracts from the invasive Argentine Ant ( Linepithema humile ) and the osm-9 ion channel is required for this response. Divergent strains varied in their response to L. humile extracts, suggesting genetic variation underlying chemotactic responses. These experiments were conducted by an undergraduate laboratory course, highlighting how C. elegans chemotaxis assays in a classroom setting can provide genuine research experiences and reveal new insights into interspecies interactions.
RESUMEN
Encircling and traversing the cell are architectural struts and dynamic intracellular highways made of cylindrical polymers called microtubules. Built from structurally asymmetric subunits of αß-tubulin heterodimers, microtubules have an inherent structural polarity with a slow-growing minus end and a comparatively dynamic plus end that grows and shrinks. Thus, a key feature of microtubules is that each polymer is polarized, allowing for the execution of cellular tasks that are directional in nature. For example, microtubules build polarized highways allowing directional intracellular transport, generate directional force such as in chromosome alignment and segregation, provide structural support for cell shape, and assemble into highly ordered polar structures like centrioles and cilia. The output of these microtubule-based functions is the performance of different tasks, including establishment and maintenance of cellular polarity, secretion and absorption, cell-cell communication, migration, mechanical resiliency, and mitosis. Different cells accomplish these functions by using distinct sites within the cell called microtubule-organizing centers (MTOCs) to build cell-specific microtubule arrangements. While the specific requirement for microtubules in many in vivo cell types is unknown, disrupting even a subset of microtubule-supported functions is often lethal and is associated with many diseases (e.g., cancer and neuropathies), suggesting that specific patterns of microtubule organization are likely important for cellular function in vivo. This Primer focuses on how differentiated animal and plant cells use distinct MTOCs to generate specific microtubule arrangements, how those arrangements support cellular functions, and how cells rearrange their microtubules to accommodate changing cellular tasks.
Asunto(s)
Centro Organizador de los Microtúbulos , Tubulina (Proteína) , Animales , Centriolos , Microtúbulos , MitosisRESUMEN
Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts.
All living things are made up of cells that form the different tissues, organs and structures of an organism. The human body, for example, is thought to consist of some 37 trillion cells and harbor over 200 cell types. To maintain a working organism, cells divide to create new cells and replace the ones that have died. Cell division is a tightly controlled process consisting of several steps, and cells continuously face a Shakespearean dilemma of deciding whether to continue dividing (also known as cell proliferation) or to halt the process (known as quiescence). This difficult balancing act is critical during all stages of life, from embryonic development to tissue growth in an adult. Problems in the underlying pathways can result in diseases such as cancer. Cell division is driven by proteins called CDKs, which help cells to complete their cell cycle in the correct sequence. To gain more insight into this complex process, scientists have developed tools for monitoring CDKs. One such tool is a fluorescent biosensor, a molecule that can be inserted into cells that glows and moves in response to CDK activity. The biosensor can be studied and measured in each cell using a microscope. Adikes, Kohrman, Martinez et al. adapted and optimized an existing CDK biosensor to help study cell division and the switch between proliferation and quiescence in two common research organisms, the nematode Caenorhabditis elegans and the zebrafish. Analysis of this biosensor showed that CDK activity at the end of cell division is higher if the cells will divide again but is low if the cells are going to become quiescent. This could suggest that the decision of a cell between proliferation and quiescence may happen earlier than expected. The optimized biosensor is sensitive enough to detect these differences and can even measure variations that influence proliferation in a region on C. elegans that was once thought to be unchanging. The development of this biosensor provides a useful research tool that could be used in other living organisms. Many research questions relate to cell division and so the applications of this tool are wide ranging.
Asunto(s)
Técnicas Biosensibles/métodos , Caenorhabditis elegans/citología , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular/fisiología , División Celular , Proliferación Celular/fisiología , Quinasas Ciclina-Dependientes/metabolismoRESUMEN
How sexually dimorphic gonads are generated is a fundamental question at the interface of developmental and evolutionary biology [1-3]. In C. elegans, sexual dimorphism in gonad form and function largely originates in different apportionment of roles to three regulatory cells of the somatic gonad primordium in young larvae. Their essential roles include leading gonad arm outgrowth, serving as the germline niche, connecting to epithelial openings, and organizing reproductive organ development. The development and function of the regulatory cells in both sexes requires the basic-helix-loop-helix (bHLH) transcription factor HLH-2, the sole ortholog of the E proteins mammalian E2A and Drosophila Daughterless [4-8], yet how they adopt different fates to execute their different roles has been unknown. Here, we show that each regulatory cell expresses a distinct complement of bHLH-encoding genes-and therefore distinct HLH-2:bHLH dimers-and formulate a "bHLH code" hypothesis for regulatory cell identity. We support this hypothesis by showing that the bHLH gene complement is both necessary and sufficient to confer particular regulatory cell fates. Strikingly, prospective regulatory cells can be directly reprogrammed into other regulatory cell types simply by loss or ectopic expression of bHLH genes, and male-to-female and female-to-male transformations indicate that the code is instructive for sexual dimorphism. The bHLH code appears to be embedded in a bow-tie regulatory architecture [9, 10], wherein sexual, positional, temporal, and lineage inputs connect through bHLH genes to diverse outputs for terminal features and provides a plausible mechanism for the evolutionary plasticity of gonad form seen in nematodes [11-15].
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Gónadas/crecimiento & desarrollo , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Caracteres SexualesRESUMEN
The prospective ventral uterus of the hermaphrodite gonad primordium consists of two pairs of sister cells, with each pair consisting of a proximal "α" cell and a distal "ß" cell. All four cells initially are competent to become the anchor cell (AC), a unique cell type that acts as the organizer of subsequent uterine and vulval development. However, the ß cells soon lose this competence and always become ventral uterine precursor cells (VUs), whereas the α cells maintain their AC competence longer, until lin-12/Notch-mediated interactions between them specify one as the AC and the other as a VU. Here, we investigate this asymmetry in developmental potential and VU fate specification between the α and ß sister cells. We find evidence that lin-12 activity contributes to the robustness of ßVU fate at elevated temperature, that the Caenorhabditis elegans Notch paralog glp-1 is not functionally redundant with lin-12 in specifying ßVU fate, and that the activity of POP-1, the sole C. elegans TCF ortholog, influences ßVU fate. We propose a model for how Wnt and LIN-12/Notch signaling together lead to robust specification of the ßVU fate.