RESUMEN
Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons. We show that impaired presynaptic actin assembly in the near absence of mDia1/3 and reduced RhoA activity is partly compensated by hyperactivation of Rac1. Inhibition of Rac1 signaling further aggravates impaired presynaptic endocytosis elicited by loss of mDia1/3. Our data suggest that interdependent mDia1/3-Rho and Rac1 signaling pathways cooperatively act to facilitate synaptic vesicle endocytosis by controlling presynaptic F-actin.
Asunto(s)
Actinas , Proteínas de Unión al GTP rho , Animales , Ratones , Transducción de Señal , Transmisión Sináptica , EndocitosisRESUMEN
Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex multilayered proteome with partially amorphous nature. Here we used crosslinking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation, bridging nucleocapsid to viral envelope. We observed the layer-specific incorporation of 82 host proteins, of which 39 are selectively recruited. We uncovered how UL32, by recruitment of PP-1 phosphatase, antagonizes binding to 14-3-3 proteins. This mechanism assures effective viral biogenesis, suggesting a perturbing role of UL32-14-3-3 interaction. Finally, we integrated these data into a coarse-grained model to provide global insights into the native configuration of virus and host protein interactions inside herpesvirions.
Asunto(s)
Citomegalovirus , Virión , Humanos , Citomegalovirus/metabolismo , Virión/metabolismo , Proteínas Virales/metabolismo , Nucleocápside/metabolismo , ProteomaRESUMEN
Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. Our data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis.
Asunto(s)
Transporte Axonal/fisiología , Lisosomas/metabolismo , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Drosophila/metabolismo , Hipocampo/metabolismo , Ratones , Neuronas/metabolismo , Transporte de Proteínas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Loss of the lysosomal ClC-7/Ostm1 2Cl(-)/H(+) exchanger causes lysosomal storage disease and osteopetrosis in humans and additionally changes fur colour in mice. Its conversion into a Cl(-) conductance in Clcn7(unc/unc) mice entails similarly severe lysosomal storage, but less severe osteopetrosis and no change in fur colour. To elucidate the basis for these phenotypical differences, we generated Clcn7(td/td) mice expressing an ion transport-deficient mutant. Their osteopetrosis was as severe as in Clcn7(-/-) mice, suggesting that the electric shunt provided by ClC-7(unc) can partially rescue osteoclast function. The normal coat colour of Clcn7(td/td) mice and their less severe neurodegeneration suggested that the ClC-7 protein, even when lacking measurable ion transport activity, is sufficient for hair pigmentation and that the conductance of ClC-7(unc) is harmful for neurons. Our in vivo structure-function analysis of ClC-7 reveals that both protein-protein interactions and ion transport must be considered in the pathogenesis of ClC-7-related diseases.
Asunto(s)
Proteínas de la Membrana/metabolismo , Alelos , Animales , Transporte Biológico , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Fibroblastos/metabolismo , Genotipo , Color del Cabello/genética , Homeostasis , Iones/metabolismo , Lisosomas/metabolismo , Melanocitos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Osteopetrosis/genética , Osteopetrosis/metabolismo , Osteopetrosis/patología , Fenotipo , Vía de Señalización WntRESUMEN
Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.
Asunto(s)
Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Moléculas de Adhesión Celular/metabolismo , Canales de Cloruro/metabolismo , Leucoencefalopatías/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/ultraestructura , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Canales de Cloruro CLC-2 , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular Neurona-Glia/genética , Cerebelo/metabolismo , Cerebelo/patología , Canales de Cloruro/genética , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Células HeLa , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/patología , Potenciales de la Membrana , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Proteínas del Tejido Nervioso/genética , Oligodendroglía/metabolismo , Oligodendroglía/patología , Oligodendroglía/ultraestructura , Técnicas de Placa-ClampRESUMEN
Excellent methods exist to analyze sub-neuronal structures, such as synapses, at nanometer resolution with electron microscopy. However, due to methodological constraints, electron microscopy is feasible only for small volumes of fixed tissue. By contrast, confocal or two-photon laser scanning microscopy is well suited to obtain neuronal structures from large volumes of living or fixed tissue at sub-micrometer resolution. Therefore, a gap exists when analyzing synaptic organization of neuropils, or the distribution of synapses throughout the dendritic trees of individual neurons, and it would be advantageous to use confocal microscopy to investigate the synaptic organization of central neuropils. This study uses correlative electron and confocal microscopy from the same tissue sections to test whether synapsin I-immunopositive puncta can be analyzed at the light microscopy level to estimate the distributions of synaptic sites within central motor neuropils and along reconstructed dendritic surfaces in an insect ventral nerve cord. It demonstrates that every type 1 synaptic terminal can be detected as a distinct punctum by synapsin I-immunolabeling and confocal microscopy. Furthermore, it provides data indicating that co-localization analysis from confocal image stacks as recently published provides a good estimate for quantifying the distribution patterns of input synapses through dendritic trees.