RESUMO
The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.
Assuntos
Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Síndrome de Wiskott-Aldrich/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto de Actina/genética , Proteína 2 Relacionada a Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Apoptose/genética , Citocromos c/genética , Dano ao DNA/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Interferente Pequeno/genética , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.
Assuntos
Actinas , Autofagossomos , Autofagia , Rim , Camundongos Knockout , Animais , Camundongos , Actinas/metabolismo , Autofagia/fisiologia , Humanos , Autofagossomos/metabolismo , Rim/metabolismo , Masculino , Túbulos Renais Proximais/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Polimerização , Fibroblastos/metabolismoRESUMO
The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.
RESUMO
The bacteriophage Lambda (λ) "Red" recombination system has enabled the development of efficient methods for engineering bacterial chromosomes. This system has been particularly important to the field of bacterial pathogenesis, where it has advanced the study of virulence factors from Shiga toxin-producing and enteropathogenic Escherichia coli (STEC and EPEC). Transient plasmid-driven expression of Lambda Red allows homologous recombination between PCR-derived linear DNA substrates and target loci in the STEC/EPEC chromosomes. Red-associated techniques can be used to create individual gene knockouts, generate deletions of large pathogenicity islands, and make markerless allelic exchanges. This chapter describes specific strategies and procedures for performing Lambda Red-mediated genome engineering in STEC.
Assuntos
Bacteriófago lambda/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Recombinação Genética , Escherichia coli Shiga Toxigênica/metabolismo , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Escherichia coli Enteropatogênica/genética , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Escherichia coli Shiga Toxigênica/genética , Proteínas Virais/genéticaRESUMO
Actin nucleation factors function to organize, shape, and move membrane-bound organelles, yet they remain poorly defined in relation to disease. Galloway-Mowat syndrome (GMS) is an inherited disorder characterized by microcephaly and nephrosis resulting from mutations in the WDR73 gene. This core clinical phenotype appears frequently in the Amish, where virtually all affected individuals harbor homozygous founder mutations in WDR73 as well as the closely linked WHAMM gene, which encodes a nucleation factor. Here we show that patient cells with both mutations exhibit cytoskeletal irregularities and severe defects in autophagy. Reintroduction of wild-type WHAMM restored autophagosomal biogenesis to patient cells, while inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and clearance of ubiquitinated protein aggregates. Normal WHAMM function involved binding to the phospholipid PI(3)P and promoting actin nucleation at nascent autophagosomes. These results reveal a cytoskeletal pathway controlling autophagosomal remodeling and illustrate several molecular processes that are perturbed in Amish GMS patients.