RESUMEN
Even the simplest cells show a remarkable degree of intracellular patterning. Like developing multicellular organisms, single cells break symmetry to establish polarity axes, pattern their cortex and interior, and undergo morphogenesis to acquire sometimes complex shapes. Symmetry-breaking and molecular patterns can be established through coupling of negative and positive feedback reactions in biochemical reaction-diffusion systems. Physical forces, perhaps best studied in the contraction of the metazoan acto-myosin cortex, which induces cortical and cytoplasmic flows, also serve to pattern-associated components. A less investigated physical perturbation is the in-plane flow of plasma membrane material caused by membrane trafficking. In this review, we discuss how bulk membrane flows can be generated at sites of active polarized secretion and growth, how they affect the distribution of membrane-associated proteins, and how they may be harnessed for patterning and directional movement in cells across the tree of life.
Asunto(s)
Polaridad Celular , Miosinas , Animales , Membrana Celular/metabolismo , Miosinas/metabolismo , Citoplasma/metabolismo , Proteínas de la MembranaRESUMEN
Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membraneassociated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.
RESUMEN
The fission yeast cells Schizosaccharomyces pombe divide at constant cell size regulated by environmental stimuli. An important pathway of cell size control involves the membrane-associated DYRK-family kinase Pom1, which forms decreasing concentration gradients from cell poles and inhibits mitotic inducers at midcell. Here, we identify the phosphatase 2C Ptc1 as negative regulator of Pom1. Ptc1 localizes to cell poles in a manner dependent on polarity and cell-wall integrity factors. We show that Ptc1 directly binds Pom1 and can dephosphorylate it in vitro but modulates Pom1 localization indirectly upon growth in low-glucose conditions by influencing microtubule stability. Thus, Ptc1 phosphatase plays both direct and indirect roles in the Pom1 cell size control pathway.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2C/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Tamaño de la Célula , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis , Fosforilación , Proteínas Quinasas/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Protein concentration gradients pattern developing organisms and single cells. In Schizosaccharomyces pombe rod-shaped cells, Pom1 kinase forms gradients with maxima at cell poles. Pom1 controls the timing of mitotic entry by inhibiting Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Using quantitative PALM imaging, we find individual Pom1 molecules bind the membrane too transiently to diffuse from pole to mid-cell. Instead, we propose they exchange within longer lived clusters forming the functional gradient unit. An allelic series blocking auto-phosphorylation shows that multi-phosphorylation shapes and buffers the gradient to control mid-cell levels, which represent the critical Cdr2-regulating pool. TIRF imaging of this cortical pool demonstrates more Pom1 overlaps with Cdr2 in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. Thus, the gradients modulate Pom1 mid-cell levels according to cell size.
Asunto(s)
Citoplasma/enzimología , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Membrana Celular/metabolismo , Fosforilación , Unión Proteica , Proteínas Quinasas/análisis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/análisisRESUMEN
All cells show size homeostasis owing to coordination of division with growth. In this issue, Allard et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201709171) establish that transient inhibitory visits of a negative regulator of Cdk1 to cortical oligomeric platforms increase in number and duration with cell growth, suggesting how Cdk1 activation is coupled to cell size.
Asunto(s)
Tamaño de la Célula , Schizosaccharomyces/citología , Mitosis , Modelos Biológicos , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The extent to which chromosomal gene position in prokaryotes affects local gene expression remains an open question. Several studies have shown that chromosomal re-positioning of bacterial transcription units does not alter their expression pattern, except for a general decrease in gene expression levels from chromosomal origin to terminus proximal positions, which is believed to result from gene dosage effects. Surprisingly, the question as to whether this chromosomal context independence is a cis encoded property of a bacterial transcription unit, or if position independence is a property conferred by factors acting in trans, has not been addressed so far. For this purpose, we established a genetic test system assessing the chromosomal positioning effects by means of identical promoter-fluorescent reporter gene fusions inserted equidistantly from OriC into both chromosomal replichores of Escherichia coli K-12. Our investigations of the reporter activities in mutant cells lacking the conserved nucleoid associated protein HU uncovered various drastic chromosomal positional effects on gene transcription. In addition we present evidence that these positional effects are caused by transcriptional activity nearby the insertion site of our reporter modules. We therefore suggest that the nucleoid-associated protein HU is functionally insulating transcription units, most likely by constraining transcription induced DNA supercoiling.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Transcripción Genética/fisiología , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Complejo de Reconocimiento del Origen/fisiología , Regiones Promotoras Genéticas/fisiologíaRESUMEN
Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.
Asunto(s)
Posicionamiento de Cromosoma , Cromosomas Bacterianos , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/genética , Regulación Bacteriana de la Expresión Génica , Antibacterianos/farmacología , ADN Superhelicoidal/análisis , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/crecimiento & desarrollo , Proteínas de Escherichia coli/biosíntesis , Factor Proteico para Inverción de Estimulación/biosíntesis , Genes Reguladores , Operón , Estrés Oxidativo , FenotipoRESUMEN
The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , Proteínas de Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/genética , Regiones Promotoras Genéticas , Iniciación de la Transcripción Genética , ADN Bacteriano/genética , ADN Superhelicoidal/genética , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Microscopía de Fuerza Atómica , Unión ProteicaRESUMEN
Cysteine cathepsins are expressed in most tissues, including the gastrointestinal tract. We demonstrated an involvement of mouse intestinal cathepsin B in extracellular matrix remodeling for regeneration from trauma. The present study aimed at elucidating roles of cysteine cathepsins in the non-traumatized gastrointestinal tract of mice. Thus we investigated expression and localization patterns of cathepsin B and its closest relative, cathepsin X, along the length of the gastrointestinal tract, and determined the effects of their absence. Cathepsin B showed the highest protein levels in the anterior segments of the gastrointestinal tract, whereas the highest activity was observed in the jejunum, as revealed by cathepsin B-specific activity-based probe labeling. Cathepsin X was most abundant in the jejunum and protein levels were elevated in duodenum and colon of Ctsb-/- mice. The segmental pattern of cathepsin expression was reflected by a compartmentalized distribution of junction proteins and basal lamina constituents, changes in tissue architecture and altered activities of the brush border enzyme aminopeptidase N. In conclusion, we observed different compensatory effects and activity levels of cysteine peptidases along the length of the small and large intestines in a segment-specific manner suggesting specific in situ functions of these enzymes in particular parts of the gastrointestinal tract.