RESUMEN
Although the properties of the actin cytoskeleton in the cytoplasm are well characterized, the regulation and function of nuclear actin filaments are only recently emerging. We previously demonstrated serum-induced, transient assembly of filamentous actin within somatic cell nuclei. However, the extracellular cues, cell surface receptors as well as underlying signaling mechanisms have been unclear. Here we demonstrate that physiological ligands for G protein-coupled receptors (GPCRs) promote nuclear F-actin assembly via heterotrimeric Gαq proteins. Signal-induced nuclear actin responses require calcium release from the endoplasmic reticulum (ER) targeting the ER-associated formin INF2 at the inner nuclear membrane (INM). Notably, calcium signaling promotes the polymerization of linear actin filaments emanating from the INM towards the nuclear interior. We show that GPCR and calcium elevations trigger nuclear actin-dependent alterations in chromatin organization, uncovering a general cellular mechanism by which physiological ligands and calcium promote nuclear F-actin assembly for rapid responses towards chromatin dynamics.
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Señalización del Calcio , Cromatina/genética , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Mitocondrias/metabolismo , Células 3T3 NIH , Polimerizacion , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.
Asunto(s)
Actinas/metabolismo , Cromatina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Animales , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Tamaño del Núcleo Celular , Ensamble y Desensamble de Cromatina/fisiología , Cofilina 1/genética , Cofilina 1/metabolismo , Fase G1/fisiología , Ratones , Mitosis/fisiología , Modelos Biológicos , Células 3T3 NIH , Optogenética , Multimerización de ProteínaRESUMEN
The Chlamydiales are an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species is Chlamydia trachomatis. Many uncertainties remain as to how Chlamydia organizes its intracellular development and replication. The discovery of new Chlamydiales species from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to "chlamydial" development. We used this approach in the infection of human cells with Waddlia chondrophila, a species from the family Waddliaceae whose natural host is uncertain. Compared to C. trachomatis, W. chondrophila had slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for the C. trachomatis inclusion. C. trachomatis infection generates proteolytic activity by the protease Chlamydia protease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case of W. chondrophila. Unlike Chlamydia, W. chondrophila did not protect against staurosporine-induced apoptosis. C. trachomatis infection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent from W. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development of Chlamydiales in vitro but may be linked to host adaptation of Chlamydia and the higher virulence of C. trachomatis.
Asunto(s)
Apoptosis , Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydiales/crecimiento & desarrollo , Aparato de Golgi/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Esfingomielinas/metabolismo , Transporte Biológico , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/fisiopatología , Chlamydia trachomatis/genética , Chlamydiales/genética , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/fisiopatología , Células HeLa , HumanosRESUMEN
Streptomyces coelicolor contains two gene clusters putatively involved in wall teichoic acid biosynthesis. Inactivation of the tagF homologue SCO2997 or SCO2584, a component of the Streptomyces spore wall synthesizing complex, affected sporulation. The mutant phenotypes resembled those of mre mutants, suggesting a function of wall teichoic acids in the differentiation of Streptomyces.
Asunto(s)
Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/genética , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/genética , Ácidos Teicoicos/metabolismo , Vías Biosintéticas/genética , Técnicas de Inactivación de Genes , Esporas Bacterianas/citología , Esporas Bacterianas/metabolismo , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismoRESUMEN
It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Familia de Multigenes , Esporas Bacterianas/metabolismo , Streptomyces coelicolor/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Pared Celular/genética , Datos de Secuencia Molecular , Esporas Bacterianas/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Streptomyces coelicolor/metabolismo , Secuencia de Aminoácidos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Transporte de Proteínas , Esporas Bacterianas/fisiología , Streptomyces coelicolor/genéticaRESUMEN
Hepatocytes of the periportal and perivenous zones of the liver lobule show marked differences in the contents and activities of many enzymes and other proteins. Previous studies from our and other groups have pointed towards an important role of beta-catenin-dependent signaling in the regulation of expression of genes encoding proteins with preferential perivenous localization, whereas, in contrast, signaling through Ras-dependent pathway(s) may induce a 'periportal' phenotype. We have now conducted a series of experiments to further investigate this hypothesis. In transgenic mice with scattered expression of an activated Ha-ras (Ha-ras(G12V)) mutant in liver, expression of the perivenous markers glutamine synthetase and two cytochrome P450 isoforms was completely abolished in those hepatocytes demonstrating constitutively activated extracellular signal-regulated kinase activity, even though they were located directly adjacent to central veins. Similarly, incubation of primary hepatocytes or hepatoma cells with increasing amounts of serum caused a concentration-dependent attenuation of expression of perivenous marker mRNAs, whereas the expression of periportal markers was increased. The inhibitory effect of high amounts of serum on the expression of perivenous markers was also observed if their expression was stimulated by activation of beta-catenin signaling, and comparable inhibitory effects were seen in cells stably transfected with a T-cell factor/lymphoid-enhancing factor-driven luciferase reporter. Epidermal growth factor could partly mimic serum effects in hepatoma cells, and its effect could be blocked by an inhibitor of extracellular signal-regulated kinase activity. These data suggest that activation of the Ras/mitogen-activated protein kinase (extracellular signal-regulated kinase) pathway favors periportal gene expression while simultaneously antagonizing a perivenous phenotype of hepatocytes.