RESUMEN
Matrix metalloproteinases (MMPs) degrade several ECM components and are crucial modulators of cell invasion and tissue organization. Although much has been reported about their function in remodeling ECM in health and disease, their trafficking across the Golgi apparatus remains poorly understood. Here we report that the cis-Golgi protein nucleobindin-1 (NUCB1) is critical for MMP2 and MT1-MMP trafficking along the Golgi apparatus. This process is Ca2+-dependent and is required for invasive MDA-MB-231 cell migration as well as for gelatin degradation in primary human macrophages. Our findings emphasize the importance of NUCB1 as an essential component of MMP transport and its overall impact on ECM remodeling.
Asunto(s)
Neoplasias de la Mama/enzimología , Matriz Extracelular/enzimología , Aparato de Golgi/enzimología , Macrófagos/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Nucleobindinas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Calcio/metabolismo , Señalización del Calcio , Movimiento Celular , Matriz Extracelular/patología , Femenino , Gelatina/metabolismo , Células HEK293 , Células HeLa , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/genética , Nucleobindinas/genética , Transporte de Proteínas , Proteolisis , Factores de TiempoRESUMEN
The TGN is a key compartment for the sorting and secretion of newly synthesized proteins. At the TGN, soluble proteins are sorted based on the instructions carried in their oligosaccharide backbones or by a Ca2+-mediated process that involves the cargo-sorting protein Cab45. Here, we show that Cab45 is phosphorylated by the Golgi-specific protein kinase Fam20C. Mimicking of phosphorylation translocates Cab45 into TGN-derived vesicles, which goes along with an increased export of LyzC, a Cab45 client. Our findings demonstrate that Fam20C plays a key role in the export of Cab45 clients by fine-tuning Cab45 oligomerization and thus impacts Cab45 retention in the TGN.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Quinasa de la Caseína I/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Transporte de Proteínas/genética , Red trans-Golgi/metabolismo , Sistemas CRISPR-Cas , Proteínas de Unión al Calcio/genética , Quinasa de la Caseína I/deficiencia , Quinasa de la Caseína I/genética , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Técnicas de Inactivación de Genes , Glicoproteínas/genética , Humanos , Isoantígenos/metabolismo , Mutación , Fosforilación , Transporte de Proteínas/fisiología , ARN Interferente Pequeño , Proteínas Recombinantes , Proteínas de Plasma Seminal/metabolismoRESUMEN
Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are segregated for their release into the extracellular space remains a challenge. We have previously demonstrated that, in a reaction that requires Ca(2+), the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. Here, we report that Cab45 reversibly assembles into oligomers in the presence of Ca(2+) These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca(2+)-dependent manner in vitro. In intact cells, mutation of the Ca(2+)-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca(2+) pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca(2+)-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN.
Asunto(s)
Señalización del Calcio , Proteínas de Unión al Calcio/fisiología , Glicoproteínas/fisiología , Red trans-Golgi/metabolismo , Transporte Biológico , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Glicoproteínas/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Proteínas/metabolismo , Vías SecretorasRESUMEN
Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization.
Asunto(s)
Actomiosina/metabolismo , Células Epiteliales/fisiología , Microvellosidades/fisiología , Miosina Tipo II/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/fisiología , Polaridad Celular/fisiología , Proliferación Celular , Perros , Epitelio/metabolismo , Células HeLa , Factor de Crecimiento de Hepatocito/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Uniones Intercelulares , Células MCF-7 , Células de Riñón Canino Madin Darby , Miosina Tipo II/antagonistas & inhibidoresRESUMEN
The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca(2+) entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca(2+) import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.
Asunto(s)
Actinas/metabolismo , Señalización del Calcio , ATPasas Transportadoras de Calcio/metabolismo , Cofilina 1/fisiología , Calcio/fisiología , ATPasas Transportadoras de Calcio/genética , Células HeLa , Humanos , Mutación Puntual , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Vías SecretorasRESUMEN
Establishment of cell polarity--or symmetry breaking--relies on local accumulation of polarity regulators. Although simple positive feedback is sufficient to drive symmetry breaking, it is highly sensitive to stochastic fluctuations typical for living cells. Here, by integrating mathematical modelling with quantitative experimental validations, we show that in the yeast Saccharomyces cerevisiae a combination of actin- and guanine nucleotide dissociation inhibitor-dependent recycling of the central polarity regulator Cdc42 is needed to establish robust cell polarity at a single site during yeast budding. The guanine nucleotide dissociation inhibitor pathway consistently generates a single-polarization site, but requires Cdc42 to cycle rapidly between its active and inactive form, and is therefore sensitive to perturbations of the GTPase cycle. Conversely, actin-mediated recycling of Cdc42 induces robust symmetry breaking but cannot restrict polarization to a single site. Our results demonstrate how cells optimize symmetry breaking through coupling between multiple feedback loops.
Asunto(s)
Polaridad Celular , Retroalimentación Fisiológica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Simulación por Computador , ADN de Hongos/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Procesos Estocásticos , Tiazolidinas/farmacología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
The plasma membrane is made up of lipids and proteins, and serves as an active interface between the cell and its environment. Many plasma-membrane proteins are laterally segregated in the plane of the membrane, but the underlying mechanisms remain controversial. Here we investigate the distribution and dynamics of a representative set of plasma-membrane-associated proteins in yeast cells. These proteins were distributed non-homogeneously in patterns ranging from distinct patches to nearly continuous networks, and these patterns were in turn strongly influenced by the lipid composition of the plasma membrane. Most proteins segregated into distinct domains. However, proteins with similar or identical transmembrane sequences (TMSs) showed a marked tendency to co-localize. Indeed we could predictably relocate proteins by swapping their TMSs. Finally, we found that the domain association of plasma-membrane proteins has an impact on their function. Our results are consistent with self-organization of biological membranes into a patchwork of coexisting domains.