RESUMO
Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow-typically at the expense of others that undergo cell death2-4. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability5. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping6. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.
RESUMO
Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.
Assuntos
Septinas/fisiologia , Vacínia/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Membrana Celular/virologia , Clatrina/análise , Clatrina/metabolismo , Dinaminas/metabolismo , Dinaminas/fisiologia , Células HeLa , Humanos , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/fisiologia , Fosforilação , Interferência de RNA , Septinas/análise , Septinas/antagonistas & inibidores , Transdução de Sinais , Liberação de Vírus/imunologiaRESUMO
During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Vaccinia virus/fisiologia , Proteínas Estruturais Virais/metabolismo , Liberação de Vírus , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Clatrina/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Vaccinia virus/química , Vaccinia virus/genética , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.
Assuntos
Autofagia , Mitocôndrias/metabolismo , Septinas/metabolismo , Shigella/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Humanos , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ligação ProteicaRESUMO
Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.