RESUMO
Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42â¢GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Polaridade Celular , Optogenética , Multimerização ProteicaRESUMO
Phospholipase A(2) activity plays key roles in generating lipid second messengers and regulates membrane topology through the generation of asymmetric lysophospholipids. In particular, the Group VIA phospholipase A(2) (GVIA-iPLA(2)) subfamily of enzymes functions independently of calcium within the cytoplasm of cells and has been implicated in numerous cellular processes, including proliferation, apoptosis, and membrane transport steps. However, mechanisms underlying the spatial and temporal regulation of these enzymes have remained mostly unexplored. Here, we examine the subset of Caenorhabditis elegans lipases that harbor a consensus motif common to members of the GVIA-iPLA(2) subfamily. Based on sequence homology, we identify IPLA-1 as the closest C. elegans homolog of human GVIA-iPLA(2) enzymes and use a combination of liposome interaction studies to demonstrate a role for acidic phospholipids in regulating GVIA-iPLA(2) function. Our studies indicate that IPLA-1 binds directly to multiple acidic phospholipids, including phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, and phosphorylated derivatives of phosphatidylinositol. Moreover, the presence of these acidic lipids dramatically elevates the specific activity of IPLA-1 in vitro. We also found that the addition of ATP and ADP promote oligomerization of IPLA-1, which probably underlies the stimulatory effect of nucleotides on its activity. We propose that membrane composition and the presence of nucleotides play key roles in recruiting and modulating GVIA-iPLA(2) activity in cells.
Assuntos
Nucleotídeos/química , Fosfolipases A2 Independentes de Cálcio/metabolismo , Fosfolipídeos/química , Animais , Caenorhabditis elegans , Calorimetria/métodos , Membrana Celular/metabolismo , Dimerização , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Genoma , Fosfolipases A2 do Grupo VI/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipossomos/química , Lipossomos/metabolismo , Mutação , Fosfolipases/metabolismo , Fosfolipases A2 Independentes de Cálcio/química , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160-166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Moduladores de Tubulina/farmacologia , Regulação para Cima/efeitos dos fármacos , Aneuploidia , Animais , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Cromossomos Humanos/metabolismo , Humanos , Cinetocoros/efeitos dos fármacos , Cinetocoros/metabolismo , Proteínas Mad2 , Camundongos , Microtúbulos/metabolismo , Modelos Biológicos , Prognóstico , Proteínas Repressoras/metabolismo , Fatores de TempoRESUMO
Export of proteins from the endoplasmic reticulum in COPII-coated vesicles occurs at defined sites that contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the endoplasmic reticulum in Caenorhabditis elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers that facilitate the co-assembly of SEC-16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a marked decline in both SEC-16 and COPII levels at endoplasmic reticulum exit sites. The sequence encoding the amino terminus of human TFG has been previously identified in chromosome translocation events involving two protein kinases, which created a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to endoplasmic reticulum exit sites, where they prematurely phosphorylate substrates during endoplasmic reticulum export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Transformação Celular Neoplásica , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Retículo Endoplasmático/metabolismo , HumanosRESUMO
Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.