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1.
Mol Biol Cell ; 35(6): ar80, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38598293

RESUMO

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/metabolismo
2.
bioRxiv ; 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38328079

RESUMO

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.

3.
PLoS Genet ; 17(4): e1009512, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33872315

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/genética
4.
Methods Mol Biol ; 2291: 145-162, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704752

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/genética
5.
J Bone Miner Res ; 36(1): 199-214, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32804442

RESUMO

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).


Assuntos
Monócitos , Osteogênese , Animais , Diferenciação Celular , Epigênese Genética , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Monócitos/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Ligante RANK/metabolismo
6.
J Bone Miner Res ; 34(6): 1155-1168, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30840779

RESUMO

This is the first study to our knowledge to report a novel mutation in the interferon regulatory factor 8 gene (IRF8G388S ) associated with multiple idiopathic tooth root resorption, a form of periodontal disease. The IRF8G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. Functional assays demonstrated that the IRF8G388S mutant promoted osteoclastogenesis and failed to inhibit NFATc1-dependent transcriptional activation when compared with IRF8WT control. Further, similar to subjects with heterozygous IRF8G388S mutation, Irf8+/- mice exhibited increased osteoclast activity in the mandibular alveolar bone surrounding molar teeth. Immunohistochemistry illustrated increased NFATc1 expression in the dentoalveolar region of Irf8-/- and Irf8+/- mice when compared with Irf8+/+ controls. Genomewide analyses revealed that IRF8 constitutively bound to regulatory regions of several thousand genes in osteoclast precursors, and genetic aberration of IRF8 significantly enhanced many osteoclast-specific transcripts. Collectively, this study delineates the critical role of IRF8 in defining osteoclast lineage and osteoclast transcriptional program, which may help in better understanding of various osteoclast-mediated disorders, including periodontal disease. © 2019 American Society for Bone and Mineral Research.


Assuntos
Predisposição Genética para Doença , Fatores Reguladores de Interferon/genética , Mutação/genética , Osteoclastos/metabolismo , Reabsorção da Raiz/genética , Transcrição Gênica , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Fatores Reguladores de Interferon/química , Fatores Reguladores de Interferon/deficiência , Interferon gama/farmacologia , Arcada Osseodentária/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Linhagem , Reabsorção da Raiz/patologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genética
7.
Mol Biol Cell ; 28(19): 2492-2507, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28720660

RESUMO

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.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Amish/genética , Mutação da Fase de Leitura , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Fosfatos de Fosfatidilinositol/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Efeito Fundador , Hérnia Hiatal/genética , Homozigoto , Humanos , Proteínas de Membrana/metabolismo , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Nefrose/genética , Fosfatos de Fosfatidilinositol/genética , Proteínas/genética , Proteínas/metabolismo
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