RESUMO
How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.
Assuntos
Membrana Celular/metabolismo , Forma Celular , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/ultraestrutura , Movimento Celular , Células HEK293 , Células HL-60 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/ultraestrutura , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Pseudópodes/genética , Pseudópodes/ultraestrutura , Transdução de Sinais , Fatores de Tempo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genéticaRESUMO
A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI:http://dx.doi.org/10.7554/eLife.01296.001.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteína Coatomer/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Subunidades Proteicas/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Transporte Biológico , Células CHO , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Fusão Celular , Proteína Coatomer/química , Cricetulus , Retículo Endoplasmático/ultraestrutura , Glicosiltransferases/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Microscopia Confocal , Subunidades Proteicas/químicaRESUMO
Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAP(f) and CLIP(f) technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.