Additional file 6: Figure S5. of The intracellular plasma membrane-connected compartment in the assembly of HIV-1 in human macrophages

Imaging a monocyte-derived macrophage (MDM) infected with HIV-1 R3A PTAP−YP− by serial block-face scanning electron microscopy (SEM). Selected sections from the serial block face SEM data set. Section numbers from the first section near the bottom of the cell (000) are indicated in the top right of the panels. Images have been segmented for the intracellular plasma membrane-connected compartment (IPMC; green) or cell surface plasma membrane (yellow). I and II show the two portions of the IPMC and the white circle indicates a narrow channel connecting IPMC portion I to the cell surface (white arrow in section 108). Towards the top of the cell, clusters of virus buds are seen between membrane protrusions at the cell surface (e.g. black arrows in section 200 and 240). Scale bar, 5 μm. (TIF 8739 kb

Additional file 5: Table S1. of The intracellular plasma membrane-connected compartment in the assembly of HIV-1 in human macrophages

Quantitation of virus buds and membrane lengths for cryosections of monocyte-derived macrophages (MDMs) infected with budding-arrested HIV mutants. MDMs from four different donors were infected with the HIV-1 R3A mutants PTAP− or PTAP−YP−, and infected MDMs located by on-grid immunolabelling. Electron microscopy images were recorded and analysed by counting the number of virus buds seen either at the cell surface or within intracellular plasma membrane-connected compartments (IPMCs). Lengths of surface or IPMC membrane within the sections were measured by tracing (see Methods). Data from all of the cells summarised in Table 1. (XLSX 46 kb

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原著和名: [記載なし]科名: = unknown採集地:採集日:採集者: 萩庭丈壽整理番号: JH049465国立科学博物館整理番号: [

Loss of NDR1/2 kinases impairs endomembrane trafficking and autophagy leading to neurodegeneration.

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases, implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models, we show that only dual loss of Ndr1/2 in neurons causes neurodegeneration. This phenotype was present when NDR kinases were deleted both during embryonic development, as well as in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein Raph1/Lpd1, as a novel NDR1/2 substrate, and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking, and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health