RESUMEN
When a cell migrates, the centrosome positions between the nucleus and the leading edge of migration via the microtubule system. The protein CrpF46 (centrosome-related protein F46) has a known role during mitosis and centrosome duplication. However, how CrpF46 efficiently regulates centrosome-related cell migration is unclear. Here, we report that knockdown of CrpF46 resulted in the disruption of microtubule arrangement, with impaired centrosomal reorientation, and slowed down cell migration. In cells that express low levels of CrpF46, stress fibers were weakened, which could be rescued by recovering Flag-CrpF46. We also found that CrpF46 interacted with non-muscle myosin high chain IIA (NMHC IIA) and that its three coiled-coil domains are pivotal for its binding to NMHC IIA. Additionally, analyses of phosphorylation of NMHC IIA and RLC (regulatory light chain) demonstrated that CrpF46 was associated with myosin IIA during filament formation. Indirect immunofluorescence images indicated that NM IIA filaments were inhibited when CrpF46 was under-expressed. Thus, CrpF46 regulates cell migration by centrosomal reorientation and altering the function of the actomyosin network by controlling specific phosphorylation of myosin.
Asunto(s)
Actomiosina/metabolismo , Autoantígenos/fisiología , Movimiento Celular , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Citoesqueleto de Actina/ultraestructura , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular Tumoral , Polaridad Celular , Centrosoma , Células HeLa , Humanos , Microtúbulos/ultraestructuraRESUMEN
The mechanisms of RNA-binding proteins (RBPs)-mediated post-transcriptional regulation of pre-existing mRNAs, which is essential for spermatogenesis, remain poorly understood. In this study, we identify that a germline-specific mitochondrial RBP AMG-1(abnormal mitochondria in germline 1), a homolog of mammalian leucine-rich PPR motif-containing protein (LRPPRC), is required for spermatogenesis in Caenorhabditis elegans. The amg-1 mutation hinders germline development without affecting somatic development and leads to the aberrant mitochondrial morphology and structure associated with mitochondrial dysfunctions specifically in the germline. We demonstrate that AMG-1 is most frequently bound to mtDNA-encoded 12S and 16S ribosomal RNA, the essential components of mitochondrial ribosomes, and that 12S rRNA expression mediated by AMG-1 is crucial for germline mitochondrial protein homeostasis. Furthermore, steroid receptor RNA activator (SRA) stem loop interacting RNA binding protein (SLRP-1), a homolog of mammalian SRA stem loop interacting RNA binding protein (SLIRP) in C. elegans, interacts with AMG-1 genetically to regulate germline development and reproductive success in C. elegans. Overall, these findings reveal the novel function of mtRBP, specifically in regulating germline development.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Masculino , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Espermatogénesis/genética , Mitocondrias/metabolismo , Proteínas de Unión al ARN/genética , Mamíferos/metabolismoRESUMEN
Spermiogenesis in nematodes is a process whereby round and quiescent spermatids differentiate into asymmetric and crawling spermatozoa. The molecular mechanism underlying this symmetry breaking remains uncharacterized. In this study, we revealed that sperm-specific Na+/K+-ATPase (NKA) is evenly distributed on the plasma membrane (PM) of Caenorhabditis elegans spermatids but is translocated to and subsequently enters the invaginated membrane of the spermatozoa cell body during sperm activation. The polarization of NKA depends on the transport of cholesterol from the PM to membranous organelles (MOs) via membrane contact sites (MCSs). The inositol 5-phosphatase CIL-1 and the MO-localized PI4P phosphatase SAC-1 may mediate PI4P metabolism to drive cholesterol countertransport via sterol/lipid transport proteins through MCSs. Furthermore, the NKA function is required for C. elegans sperm motility and reproductive success. Our data imply that the lipid dynamics mediated by MCSs might play crucial roles in the establishment of cell polarity. eGraphical abstract.
Asunto(s)
Transporte Biológico/genética , Proteínas de Caenorhabditis elegans/genética , Colesterol/genética , Esterasas/genética , Proteínas de la Membrana/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Espermatogénesis/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Colesterol/metabolismo , Masculino , Membranas Mitocondriales/metabolismo , Orgánulos/genética , Motilidad Espermática/genética , Espermátides/crecimiento & desarrollo , Espermatozoides/crecimiento & desarrolloRESUMEN
INMAP is a spindle protein that plays essential role for mitosis, by ensuring spindle and centromere integrality. The aim of this study was to investigate the relevant functions of INMAP for genomic stability and its functional pathway. We overexpressed INMAP in HeLa cells, resulting in growth inhibition in monolayer cell cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice. In this system caused micronuclei (MNi) formation, chromosome distortion and γH2AX expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a tumour biochemical marker, lactate dehydrogenase (LDH) isoenzymes were detected to evaluate cell metabolic activity, the results confirming that total activity of LDH, as well as that of its LDH5 isoform, is significantly decreased in INMAP-overexpressing HeLa cells. The levels of p53 and p21 were upregulated, and however, that of PCNA and Bcl-2, downregulated. Indirect immunofluorescence (IIF) and coimmunoprecipitation (CoIP) analyses revealed the interaction between INMAP and p21. These results suggest that INMAP might function through p53/p21 pathways.