RESUMO
The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation.
Assuntos
Actinas , Mitocôndrias , Profilinas , Profilinas/metabolismo , Profilinas/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Humanos , Actinas/metabolismo , Mitofagia/genética , Lisossomos/metabolismo , Citosol/metabolismo , Técnicas de Inativação de Genes , Autofagossomos/metabolismo , Células HeLaRESUMO
RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3 Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates.
Assuntos
Genes de Plantas , Splicing de RNA , RNA Nuclear Pequeno , Zea mays/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência Conservada , Endosperma/genética , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Íntrons , Mutação , Motivos de Nucleotídeos , Fosforilação , Matrizes de Pontuação de Posição Específica , Transporte Proteico , Isoformas de RNA , Sítios de Splice de RNA , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Zea mays/metabolismoRESUMO
GPCRs are ubiquitous in mammalian cells and present intricate mechanisms for cellular signaling and communication. Mechanistically, GPCR signaling was identified to occur vectorially through heterotrimeric G proteins that are negatively regulated by GRK and arrestin effectors. Emerging evidence highlights additional roles for GRK and Arrestin partners, and establishes the existence of interconnected feedback pathways that collectively define GPCR signaling. GPCRs influence cellular dynamics and can mediate pathologic development, such as cancer and cardiovascular remolding. Hence, a better understanding of their overall signal regulation is of great translational interest and research continues to exploit the pharmacologic potential for modulating their activity.
Assuntos
Arrestinas/metabolismo , Retroalimentação Fisiológica , Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Humanos , Modelos BiológicosRESUMO
In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.
Assuntos
Actinas , Microtúbulos , Profilinas , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actinas/genética , Actomiosina/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Profilinas/metabolismo , Profilinas/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genéticaRESUMO
Microtubules, intermediate filaments, and actin are cytoskeletal polymer networks found within the cell. While each has unique functions, all the cytoskeletal elements must work together for cellular mechanics to be fully operative. This is achieved through crosstalk mechanisms whereby the different networks influence each other through signaling pathways and direct interactions. Because crosstalk can be complex, it is possible for perturbations in one cytoskeletal element to affect the others in ways that are difficult to predict. Here we investigated how long-term changes to the actin cytoskeleton affect microtubules and intermediate filaments. Reducing F-actin or actomyosin contractility increased acetylated microtubules and intermediate filament expression, with the effect being significantly more pronounced in neuronal processes. Changes to microtubules were completely reversible if F-actin and myosin activity is restored. Moreover, the altered microtubules in neuronal processes resulting from F-actin depletion caused significant changes to microtubule-based transport, mimicking phenotypes that are linked to neurodegenerative disease. Thus, defects in actin dynamics cause a compensatory response in other cytoskeleton components which profoundly alters cellular function.
RESUMO
Profilin 1 (PFN1) is an actin binding protein that is vital for the polymerization of monomeric actin into filaments. Here we screened knockout cells for novel functions of PFN1 and discovered that mitophagy, a type of selective autophagy that removes defective or damaged mitochondria from the cell, was significantly upregulated in the absence of PFN1. Despite successful autophagosome formation and fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells still accumulate damaged, dysfunctional mitochondria. Subsequent imaging and functional assays showed that loss of PFN1 significantly affects mitochondria morphology, dynamics, and respiration. Further experiments revealed that PFN1 is located to the mitochondria matrix and is likely regulating mitochondria function from within rather than through polymerizing actin at the mitochondria surface. Finally, PFN1 mutants associated with amyotrophic lateral sclerosis (ALS) fail to rescue PFN1 knockout mitochondrial phenotypes and form aggregates within mitochondria, further perturbing them. Together, these results suggest a novel function for PFN1 in regulating mitochondria and identify a potential pathogenic mechanism of ALS-linked PFN1 variants.
RESUMO
Progression of prostate cancer (PC) to terminal castration-resistant PC (CRPC) involves a diverse set of intermediates, and androgen receptor (AR) is the key mediator of PC initiation and progression to CRPC. Hence, identification of factors involved in the regulation of AR expression and function is a necessary first-step to improve disease outcome. In this study, we identified ubiquitous ßArrestin 1 (ßArr1) as a regulator of AR function in CRPC. Unbiased gene expression analysis of public datasets revealed increased levels of ARRB1 (the gene encoding ßArr1) in CRPC when compared to normal tissue. Further, ßArr1 expression correlated with enhanced AR transcriptional function in these datasets. The ßArr1 partitions to both nucleus and cytosol and mechanistic studies showed that nuclear, and not cytosolic, ßArr1 formed a complex with AR and AR-coregulator ßCatenin and that the heterotrimeric protein complex was recruited to androgen-response elements of AR-regulated genes. Functionally, we demonstrate that depletion of ßArr1 attenuates PC cell and tumor growth and metastasis, and rescued expression of nuclear, but not cytosolic, ßArr1 restores the PC colony growth and invasion of Matrigel in vitro and tumor growth and metastasis in mice. The targeting of ßArr1-regulated AR transcriptional function may be used in the development of new drugs to treat lethal CRPC.
Assuntos
Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , beta-Arrestina 1/metabolismo , Animais , Progressão da Doença , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias de Próstata Resistentes à Castração/patologia , beta-Arrestina 1/genéticaRESUMO
Treatment options for metastatic renal cell carcinoma (RCC) are limited. In this study, we investigated impact of prostaglandin E2 (PGE2) receptor 4 (EP4) on RCC metastasis. We found that knockdown of EP4 in two RCC cell lines, ACHN and SN12C, does not affect xenograft tumor take or growth rate in mice, but reduces metastasis by decreasing tumor intravasation. Using chick chorioallantoic membrane (CAM) assay, we confirmed that blockade of EP4 signaling inhibits tumor intravasation. In vitro studies associated EP4 expression and activity with RCC cell transendothelial migration (TEM). Gene expression analysis and validation assays showed that EP4 knockdown decreases expression of CD24, a ligand to the adhesion molecule P-selectin. Forced expression of CD24 in EP4 knockdown RCC rescues TEM capacity of the cells. Pharmacologic inhibition or knockdown of endothelial P-selectin blocks EP4-mediated cancer cell TEM, and inhibition of P-selectin prevents RCC tumor intravasation in CAM assay. Our results demonstrate that inhibition of EP4 attenuates the RCC intravasation and metastasis by downregulating CD24 and that P-selectin participates in tumor intravasation, implying a potential for these molecules as therapeutic targets for advanced RCC treatment.