RESUMEN
Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.
Asunto(s)
Dineínas , Proteínas de Unión al GTP rab , Dineínas/genética , Dineínas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps â¼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Emparejamiento Base , ADN/metabolismo , Daño del ADN , Reparación del ADN por Unión de Extremidades , Células HeLa , Humanos , Lisina/metabolismo , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Complejo Shelterina , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Recent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and the study of rare tumorigenic events in vivo The adult mammary gland provides a unique model for investigating physiological or pathological processes such as morphogenesis or epithelial cell dissemination. Here, we establish a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allows us to analyze the behavior of individually labeled cells in regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing atypical protein kinase C iota (also known as PRKCI, referred to here as aPKCι) with an N-terminal eGFP fusion (GFP-aPKCι+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCι+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.
Asunto(s)
Glándulas Mamarias Humanas , Organoides , Animales , Células Epiteliales , Epitelio , Humanos , Glándulas Mamarias Animales , MorfogénesisRESUMEN
The human neocortex has undergone strong evolutionary expansion, largely due to an increased progenitor population, the basal radial glial cells. These cells are responsible for the production of a diversity of cell types, but the successive cell fate decisions taken by individual progenitors remain unknown. Here we developed a semi-automated live/fixed correlative imaging method to map basal radial glial cell division modes in early fetal tissue and cerebral organoids. Through the live analysis of hundreds of dividing progenitors, we show that basal radial glial cells undergo abundant symmetric amplifying divisions, and frequent self-consuming direct neurogenic divisions, bypassing intermediate progenitors. These direct neurogenic divisions are more abundant in the upper part of the subventricular zone. We furthermore demonstrate asymmetric Notch activation in the self-renewing daughter cells, independently of basal fibre inheritance. Our results reveal a remarkable conservation of fate decisions in cerebral organoids, supporting their value as models of early human neurogenesis.
Asunto(s)
Linaje de la Célula , Neocórtex , Células-Madre Neurales , Neurogénesis , Organoides , Humanos , Neocórtex/citología , Neocórtex/embriología , Neocórtex/metabolismo , Organoides/citología , Organoides/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Diferenciación Celular , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , División Celular , Proliferación CelularRESUMEN
Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Toxina Shiga/metabolismo , Toxina Shiga/farmacología , Animales , Endosomas/química , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HeLa , Humanos , Liposomas/química , Liposomas/metabolismo , Ratones , Transporte de Proteínas/efectos de los fármacos , Shigella dysenteriaeRESUMEN
We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.
Asunto(s)
Separación Celular/métodos , Imagenología Tridimensional/métodos , Magnetismo , Microfluídica/métodos , Modelos Teóricos , Algoritmos , Línea Celular Tumoral , Separación Celular/instrumentación , Citometría de Flujo , Humanos , Inmunofenotipificación , Células Jurkat , Microfluídica/instrumentación , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Electrónica de Rastreo , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Aparato de Golgi/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Proteínas del Citoesqueleto/metabolismo , Aparato de Golgi/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Sustancias Macromoleculares/metabolismo , Transducción de Señal/fisiología , Factores de Tiempo , Proteína de Unión al GTP rhoARESUMEN
Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.
Asunto(s)
Células Ependimogliales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Animales , Linaje de la Célula , Movimiento Celular , Edad Gestacional , Humanos , Ratones , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Neocórtex/embriología , Transducción de Señal , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Polyploidy arises from the gain of complete chromosome sets [1], and it is known to promote cancer genome evolution. Recent evidence suggests that a large proportion of human tumors experience whole-genome duplications (WGDs), which might favor the generation of highly abnormal karyotypes within a short time frame, rather than in a stepwise manner [2-6]. However, the molecular mechanisms linking whole-genome duplication to genetic instability remain poorly understood. Using repeated cytokinesis failure to induce polyploidization of Drosophila neural stem cells (NSCs) (also called neuroblasts [NBs]), we investigated the consequences of polyploidy in vivo. Surprisingly, we found that DNA damage is generated in a subset of nuclei of polyploid NBs during mitosis. Importantly, our observations in flies were confirmed in mouse NSCs (mNSCs) and human cancer cells after acute cytokinesis inhibition. Interestingly, DNA damage occurs in nuclei that were not ready to enter mitosis but were forced to do so when exposed to the mitotic environment of neighboring nuclei within the same cell. Additionally, we found that polyploid cells are cell-cycle asynchronous and forcing cell-cycle synchronization was sufficient to lower the levels of DNA damage generated during mitosis. Overall, this work supports a model in which DNA damage at mitotic entry can generate DNA structural abnormalities that might contribute to the onset of genetic instability.
Asunto(s)
Ciclo Celular/fisiología , Citocinesis/genética , Daño del ADN/genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Citocinesis/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitosis/genética , Células-Madre Neurales/metabolismo , PoliploidíaRESUMEN
mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe. In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly. It compacts before anaphase into a tight ring with other cytokinetic ring components. We show here that mid1p binds to the medial cortex by at least two independent means. First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer. This association is stabilized by the polybasic NLS. mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex. Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position. Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings. In addition, we show that mid1p oligomerizes. We propose that membrane-bound oligomers of mid1p assemble recruitment "platforms" for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction.
Asunto(s)
Citocinesis , Proteínas Fúngicas/metabolismo , Membranas/metabolismo , Mitosis , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fraccionamiento Celular , Núcleo Celular/química , Proteínas Fúngicas/química , Glutatión Transferasa/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microscopía Fluorescente , Microscopía por Video , Modelos Biológicos , Mutación , Señales de Localización Nuclear , Fotoblanqueo , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Factores de TiempoRESUMEN
Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Δpkl1Δ spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Δpkl1Δ spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics-kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors.
Asunto(s)
Segregación Cromosómica/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Mitosis/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/fisiología , Simulación por Computador , Dineínas/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Modelos Biológicos , Método de Montecarlo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismoRESUMEN
The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.
Asunto(s)
Aparato de Golgi/metabolismo , Cinesinas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Vesículas Citoplasmáticas/metabolismo , Humanos , Microtúbulos/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Ratas , Red trans-Golgi/metabolismoRESUMEN
The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.
Asunto(s)
Flavivirus/fisiología , Neocórtex/citología , Neocórtex/virología , Células-Madre Neurales/virología , Tropismo Viral , Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Apoptosis , Ciclo Celular , Modelos Animales de Enfermedad , Flavivirus/clasificación , Ratones , Filogenia , Células VeroRESUMEN
Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors.
Asunto(s)
Núcleo Celular/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transporte de Proteínas , Schizosaccharomyces/ultraestructuraRESUMEN
The microtubule cytoskeleton plays important roles in cell polarity, motility and division. Microtubules inherently undergo dynamic instability, stochastically switching between phases of growth and shrinkage. In cells, some microtubule-associated proteins (MAPs) and molecular motors can further modulate microtubule dynamics. We present here the fission yeast mtr1(+), a new regulator of microtubule dynamics that appears to be not a MAP or a motor. mtr1-deletion (mtr1Δ) primarily results in longer microtubule dwell-time at the cell tip cortex, suggesting that mtr1p acts directly or indirectly as a destabilizer of microtubules. mtr1p is antagonistic to mal3p, the ortholog of mammalian EB1, which stabilizes microtubules. mal3Δ results in short microtubules, but can be partially rescued by mtr1Δ, as the double mutant mal3Δ mtr1Δ exhibits longer microtubules than mal3Δ single mutant. By sequence homology, mtr1p is predicted to be a component of the ribosomal quality control complex. Intriguingly, deletion of a predicted ribosomal gene, rps1801, also resulted in longer microtubule dwell-time similar to mtr1Δ. The double-mutant mal3Δ rps1801Δ also exhibits longer microtubules than mal3Δ single mutant alone. Our study suggests a possible involvement of mtr1p and the ribosome complex in modulating microtubule dynamics.
RESUMEN
Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Dinaminas/química , Proteínas Nucleares/química , Fosfatidilinositoles/química , Proteínas Supresoras de Tumor/química , Secuencias de Aminoácidos , Membrana Celular/química , Endocitosis , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Células HeLa , Humanos , Membrana Dobles de Lípidos/química , Liposomas/química , Simulación de Dinámica Molecular , Músculos/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.
Asunto(s)
Citocinesis , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Segregación Cromosómica , Cromosomas Fúngicos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Fosforilación , Unión Proteica , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/químicaRESUMEN
The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers.
Asunto(s)
Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Ciclo Celular/fisiología , Células HeLa , Humanos , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Maintaining genome integrity and cellular function requires proper positioning of the cell division plane. In most eukaryotes, cytokinesis relies on a contractile actomyosin ring positioned by intrinsic spatial signals that are poorly defined at the molecular level. Fission yeast cells assemble a medial contractile ring in response to positive spatial cues from the nucleus at the cell center and negative spatial cues from the cell tips. These signals control the localization of the anillin-like protein Mid1, which defines the position of the division plane at the medial cortex, where it recruits contractile-ring components at mitosis onset. Here we show that Cdr2 kinase anchors Mid1 at the medial cortex during interphase through association with the Mid1 N terminus. This association underlies the negative regulation of Mid1 distribution by cell tips. We also demonstrate that the positive signaling from the nucleus is based on Mid1 nuclear export, which links division-plane position to nuclear position during early mitosis. After nuclear displacement, Mid1 nuclear export is dominant over Cdr2-dependent positioning of Mid1. We conclude that Cdr2- and nuclear export-dependent positioning of Mid1 constitute two overlapping mechanisms that relay cell polarity and nuclear positional information to ensure proper division-plane specification.
Asunto(s)
Proteínas Contráctiles/fisiología , Citocinesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/citología , Transporte Activo de Núcleo Celular , Bencimidazoles/farmacología , Carbamatos/farmacología , Núcleo Celular/ultraestructura , Polaridad Celular , Proteínas Contráctiles/metabolismo , Interfase/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
Recent progress in biology and microscopy has made it possible to acquire multidimensional data on rapid cellular activities. Unfortunately, the data analysis needed to describe the observed biological process still remains a major bottleneck. We here present a novel method of studying membrane trafficking by monitoring vesicular structures moving along a three-dimensional cytoskeleton network. It allows the dynamics of such structures to be qualitatively and quantitatively investigated. Our tracking method uses kymogram analysis to extract the consistent part of the temporal information and to allow the meaningful representation of vesicle dynamics. A fully automatic extension of this method, together with a statistical tool for dynamic comparisons, allows the precise analysis and comparison of overall speed distributions and directions. It can handle typical complex situations, such as a dense set of vesicles moving at various velocities, fusing and dissociating with each other or with other cell compartments. The overall approach has been characterized and validated on synthetic data. We chose the Rab6A protein, a GTPase involved in the regulation of intracellular membrane trafficking, as a molecular model. The application of our method to GFP-Rab6A stable cells acquired using fast four-dimensional deconvolution video-microscopy gives considerable cellular dynamic information unreachable using other techniques.