RESUMEN
Drosophila male germline stem cells (GSCs) reside at the tip of the testis and surround a cluster of niche cells. Decapentaplegic (Dpp) is one of the well-established ligands and has a major role in maintaining stem cells located in close proximity. However, the existence and the role of the diffusible fraction of Dpp outside of the niche have been unclear. Here, using genetically-encoded nanobodies called Morphotraps, we physically block Dpp diffusion without interfering with niche-stem cell signaling and find that a diffusible fraction of Dpp is required to ensure differentiation of GSC daughter cells, opposite of its role in maintenance of GSC in the niche. Our work provides an example in which a soluble niche ligand induces opposed cellular responses in stem cells versus in differentiating descendants to ensure spatial control of the niche. This may be a common mechanism to regulate tissue homeostasis.
Asunto(s)
Proteínas de Drosophila , Animales , Masculino , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ligandos , Diferenciación Celular/fisiología , Drosophila/metabolismo , Transducción de Señal/fisiología , Nicho de Células Madre/fisiología , Células Germinativas/metabolismo , Drosophila melanogaster/metabolismoRESUMEN
Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments. Multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants in complex image-based geometries. This approach is applied to problems in membrane surface diffusion as well as diffusion and binding in cytosolic volumes in complex cell geometries. In addition, we model diffusion and binding within phase-separated biomolecular condensates (liquid droplets). These are modeled as spherical low-affinity binding domains that also define a high viscosity medium for exchange of the free fluorescently labeled ligand with the external cytosol.
Asunto(s)
Difusión , Fluorescencia , Recuperación de Fluorescencia tras Fotoblanqueo/métodosRESUMEN
The human (h) ZIP4 is a plasma membrane transporter that functions to increase cytosolic zinc levels. hZIP4 encodes eight transmembrane domains and a large extracellular domain (ECD). This ECD is cleaved from the holo-transporter when cells are zinc-deficient. At the same time, mutations in the ECD can result in the zinc-deficiency disease Acrodermatitis enteropathica. Previously, it was shown that hZIP4's ECD is comprised of two structurally independent subdomains where contacts between the ECD monomeric units are centered at the PAL motif. These results lead to the hypothesis that ZIP4-ECD is essential to the dimerization of the holo-transporter. To test this hypothesis, we used Fluorescence Correlation Spectroscopy (FCS) to quantify the oligomeric state of full-length hZIP4 and hZIP4 lacking the ECD domain, each tagged with eGFP. Inspection of our experimental results demonstrate that both the full-length and truncated hZIP4 is a dimer when expressed in HEK293 cells. Parallel functional experiments demonstrate that the Km and Vmax for truncated and full-length hZIP4/eGFP are similar. Determining that truncated hZIP4/eGFP forms a dimer is a crucial step for understanding the function of the hZIP4-ECD, which provides more insight into how the diseases related to hZIP4 protein.
Asunto(s)
Proteínas de Transporte de Membrana , Zinc , Humanos , Células HEK293RESUMEN
Cellular and intracellular processes are inherently complex due to the large number of components and interactions, which are often nonlinear and occur at different spatiotemporal scales. Because of this complexity, mathematical modeling is increasingly used to simulate such systems and perform experiments in silico, many orders of magnitude faster than real experiments and often at a higher spatiotemporal resolution. In this article, we will focus on the generic modeling process and illustrate it with an example model of membrane lipid turnover.
Asunto(s)
Biología Celular , Modelos Biológicos , Biología Celular/estadística & datos numéricos , Biología Computacional , Simulación por Computador , Conceptos Matemáticos , Lípidos de la Membrana/metabolismo , Dinámicas no Lineales , Programas Informáticos , Análisis Espacio-TemporalRESUMEN
Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. We have previously shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. Here we demonstrate that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, our results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Nicho de Células Madre/fisiología , Células Madre/metabolismo , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiología , Animales , Diferenciación Celular/fisiología , Drosophila melanogaster/metabolismo , Células Germinativas/citología , Células Germinativas/metabolismo , Ligandos , Masculino , Microtúbulos/metabolismo , Microtúbulos/fisiología , Transducción de Señal/fisiología , Células Madre/citología , Testículo/metabolismoRESUMEN
Most computational models in biology are built and intended for "single-use"; the lack of appropriate annotation creates models where the assumptions are unknown, and model elements are not uniquely identified. Simply recreating a simulation result from a publication can be daunting; expanding models to new and more complex situations is a herculean task. As a result, new models are almost always created anew, repeating literature searches for kinetic parameters, initial conditions and modeling specifics. It is akin to building a brick house starting with a pile of clay. Here we discuss a concept for building annotated, reusable models, by starting with small well-annotated modules we call ModelBricks. Curated ModelBricks, accessible through an open database, could be used to construct new models that will inherit ModelBricks annotations and thus be easier to understand and reuse. Key features of ModelBricks include reliance on a commonly used standard language (SBML), rule-based specification describing species as a collection of uniquely identifiable molecules, association with model specific numerical parameters, and more common annotations. Physical bricks can vary substantively; likewise, to be useful the structure of ModelBricks must be highly flexible-it should encapsulate mechanisms from single reactions to multiple reactions in a complex process. Ultimately, a modeler would be able to construct large models by using multiple ModelBricks, preserving annotations and provenance of model elements, resulting in a highly annotated model. We envision the library of ModelBricks to rapidly grow from community contributions. Persistent citable references will incentivize model creators to contribute new ModelBricks.
Asunto(s)
Biología Computacional/métodos , Biología Computacional/normas , Biología de Sistemas/métodos , Simulación por Computador , Bases de Datos Factuales , Modelos Biológicos , Programas Informáticos , Biología de Sistemas/normasRESUMEN
Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the "germinosome," and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥ 75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased â¼ 50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by â¼ 2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/fisiología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Western Blotting , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismoRESUMEN
Tight junctions (TJs) feature critically in maintaining the integrity of the blood-brain barrier (BBB), and undergo significant disruption during neuroinflammatory diseases. Accordingly, the expression and distribution of CLN-5, a prominent TJ protein in central nervous system (CNS) microvessels and BBB determinant, has been shown to parallel physiological and pathophysiological changes in microvascular function. However, efforts to quantify CLN-5 within the CNS microvasculature in situ, by using conventional two-dimensional immunohistochemical analysis of thin sections, are encumbered by the tortuosity of capillaries and distorted diameters of inflamed venules. Herein, we describe a novel contour-based 3D image visualization and quantification method, employing high-resolution confocal z-stacks from thick immunofluorescently-stained thoraco-lumbar spinal cord cryosections, to analyze CLN-5 along the junctional regions of different-sized CNS microvascular segments. Analysis was performed on spinal cords of both healthy mice, and mice experiencing experimental autoimmune encephalomyelitis (EAE), an animal model of the neuroinflammatory disease multiple sclerosis. Results indicated that, under normal conditions, the density of CLN-5 staining (CLN-5 intensity/ endothelial surface area) was greatest in the capillaries and smaller venules, and least in the larger venules. This heterogeneity in junctional CLN-5 staining was exacerbated during EAE, as spinal venules revealed a significant loss of junctional CLN-5 staining that was associated with focal leukocyte extravasation, while adjacent capillaries exhibited neither CLN-5 loss nor infiltrating leukocytes. However, despite only venules displaying these behaviors, both capillaries and venules evidenced leakage of IgG during disease, further underscoring the heterogeneity of the inflammatory response in CNS microvessels. This method should be readily adaptable to analyzing other junctional proteins of the CNS and peripheral microvasculature, and serve to highlight their role(s) in health and disease.
Asunto(s)
Barrera Hematoencefálica , Claudina-5/análisis , Encefalomielitis Autoinmune Experimental/patología , Endotelio Vascular/química , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microvasos/química , Médula Espinal/irrigación sanguínea , Uniones Estrechas/química , Animales , Capilares/química , Capilares/ultraestructura , Permeabilidad Capilar , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/sangre , Encefalomielitis Autoinmune Experimental/inmunología , Endotelio Vascular/ultraestructura , Femenino , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Esclerosis Múltiple , Uniones Estrechas/ultraestructura , Vénulas/química , Vénulas/ultraestructuraRESUMEN
The shape of a cell, the sizes of subcellular compartments, and the spatial distribution of molecules within the cytoplasm can all control how molecules interact to produce a cellular behavior. This chapter describes how these spatial features can be included in mechanistic mathematical models of cell signaling. The Virtual Cell computational modeling and simulation software is used to illustrate the considerations required to build a spatial model. An explanation of how to appropriately choose between physical formulations that implicitly or explicitly account for cell geometry and between deterministic versus stochastic formulations for molecular dynamics is provided, along with a discussion of their respective strengths and weaknesses. As a first step toward constructing a spatial model, the geometry needs to be specified and associated with the molecules, reactions, and membrane flux processes of the network. Initial conditions, diffusion coefficients, velocities, and boundary conditions complete the specifications required to define the mathematics of the model. The numerical methods used to solve reaction-diffusion problems both deterministically and stochastically are then described and some guidance is provided in how to set up and run simulations. A study of cAMP signaling in neurons ends the chapter, providing an example of the insights that can be gained in interpreting experimental results through the application of spatial modeling.
Asunto(s)
Simulación por Computador , Redes y Vías Metabólicas/fisiología , Neuronas/citología , Transducción de Señal/fisiología , Programas Informáticos , Algoritmos , Animales , Forma de la Célula/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Cinética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Método de Montecarlo , Neuronas/metabolismoRESUMEN
Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990-2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.
Asunto(s)
Fagocitosis/fisiología , Fagosomas/metabolismo , ARN/aislamiento & purificación , Tetrahymena thermophila/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Bases , Eliminación de Gen , Humanos , Espectrometría de Masas , Fagosomas/química , Proteoma/análisis , Proteoma/genética , Proteoma/metabolismo , ARN/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetrahymena thermophila/metabolismoRESUMEN
Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/análisis , Membrana Celular/química , Esporas Bacterianas/química , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente , Multimerización de Proteína , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
BACKGROUND: Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides. RESULTS: A vector encoding soluble peptidylglycine alpha-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with K+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions. CONCLUSIONS: Under basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals.
Asunto(s)
Neuronas/fisiología , Vías Secretoras/fisiología , Vesículas Secretoras/fisiología , Ganglio del Trigémino/fisiología , Actinas/metabolismo , Animales , Axones/efectos de los fármacos , Axones/fisiología , Células Cultivadas , Citoesqueleto/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/fisiología , Oxigenasas de Función Mixta/metabolismo , Movimiento (Física) , Neuronas/efectos de los fármacos , Fármacos del Sistema Nervioso Periférico/farmacología , Ésteres del Forbol/farmacología , Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Vías Secretoras/efectos de los fármacos , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Factores de Tiempo , Ganglio del Trigémino/efectos de los fármacosRESUMEN
Virus-Induced Chaperone-Enriched (VICE) domains form adjacent to nuclear viral replication compartments (RC) during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70), the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/patogenicidad , Chaperonas Moleculares/fisiología , Proteínas Nucleares/fisiología , Genes Virales , Herpes Simple/prevención & control , Herpesvirus Humano 1/genética , Humanos , Proteína Huntingtina , Enfermedad de Huntington/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Pliegue de Proteína , Ubiquitina/fisiología , Proteínas Virales/fisiologíaRESUMEN
Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock > or =0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells' osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.
Asunto(s)
Bacillus subtilis/química , Bacillus subtilis/fisiología , Proteínas Bacterianas/análisis , Canales Iónicos/análisis , Mecanorreceptores/química , Presión Osmótica , Estrés Fisiológico , Fusión Artificial Génica , Western Blotting , Membrana Celular/química , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Rab3a, a small GTPase important for exocytosis, is uniquely up-regulated as oligodendrocytes enter terminal differentiation and initiate myelin biosynthesis. In this study, we analyze the role of this protein in oligodendrocyte morphological differentiation by using Rab3a overexpression and siRNAi-mediated Rab3a silencing. We found that Rab3a silencing delayed mature oligodendrocyte morphological differentiation but did not interfere with lineage progression of OL progenitors; this is consistent with the high levels of Rab3a expressed by mature oligodendrocytes compared with progenitor cells. Overexpression of GTP-bound, but not that of wild-type, Rab3a delayed OL morphological differentiation; this suggests that expression of a GTP-bound Rab3a mutant interferes with the normal function of endogenous Rab3a. We have also identified in oligodendrocytes two other exocytic small GTPases, Rab27B and RalA. Together, these findings indicate that Rab3a specifically stimulates morphological differentiation of mature oligodendrocytes and thus may be part of the necessary machinery for myelin membrane biogenesis.
Asunto(s)
Diferenciación Celular/fisiología , Oligodendroglía/citología , Células Madre/citología , Proteína de Unión al GTP rab3A/metabolismo , Animales , Western Blotting , Proteínas de Unión al GTP/metabolismo , Inmunohistoquímica , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , ARN Interferente Pequeño , Ratas , Células Madre/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/efectos de los fármacos , Hormona Luteinizante/farmacología , Meiosis/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Conexina 43/química , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/metabolismo , Femenino , Uniones Comunicantes/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Meiosis/fisiología , Ratones , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Folículo Ovárico/citología , Fosforilación , Serina/química , Técnicas de Cultivo de TejidosRESUMEN
Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.
Asunto(s)
Bacillus subtilis/química , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/análisis , Esporas Bacterianas/química , Esporas Bacterianas/ultraestructura , Aminas/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Detergentes/farmacología , Desinfectantes/farmacología , Desinfección/métodos , Electroforesis en Gel de Poliacrilamida , Alimentos , Eliminación de Gen , Calor , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía de Interferencia , Muramidasa/metabolismo , Ácidos Picolínicos/metabolismo , Hipoclorito de Sodio/farmacología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.
Asunto(s)
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Glicoproteínas de Membrana/metabolismo , Capacitación Espermática , Espermatozoides/metabolismo , Animales , Difusión , Fertilinas , Cobayas , MasculinoRESUMEN
Populations of Bacillus subtilis spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but >/=98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.
Asunto(s)
Bacillus subtilis/citología , Calor , Humedad , Esporas Bacterianas/fisiología , Centrifugación por Gradiente de Densidad , Factores de TiempoRESUMEN
A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10 degrees of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.