Cellular logistics最新文献

The phagocytosis and destruction of pathogens and dead cells by macrophages is important for innate immunity and tissue maintenance. Multiple Rab family GTPases engage effector molecules to coordinate the early stages of phagocytosis, which include rapid changes in actin polymerization, membrane phospholipids, trafficking and the activation of receptors. Defining the spatiotemporal, sequential recruitment of these Rabs is critical for insights into how phagocytosis is initiated and coordinated. Here, we screened GFP-tagged Rabs expressed in fixed and live cells to identify and stratify those recruited to early phagocytic membranes at stages defined by phospholipid transitions. We propose a sequence of Rabs 35, 13, 8a, 8b, 27a, 10, and 31 that precedes and accompanies phagocytic cup closure, followed after closure by recruitment of endosomal Rabs 5a, 5b, 5c, 14, and 11. Reducing the expression of individual Rabs by siRNA knockdown, notably Rabs 35 and 13, disrupts phagocytosis prior to phagocytic cup closure, confirming a known role for Rab35 and revealing anew the involvement of Rab13. The results enhance our understanding of innate immune responses in macrophages by revealing the sequence of Rabs that initiates phagocytosis.

Communication among neurons is mediated through synaptic connections between axons and dendrites, and most excitatory synapses occur on actin-rich protrusions along dendrites called dendritic spines. Dendritic spines are structurally dynamic, and synapse strength is closely correlated with spine morphology. Abnormalities in the size, shape, and number of dendritic spines are prevalent in neurologic diseases, including autism spectrum disorders, schizophrenia, and Alzheimer disease. However, therapeutic targets that influence spine morphology are lacking. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are potent regulators of the actin cytoskeleton and highly promising drug targets for central nervous system disorders. In this report, we addressed how pharmacologic inhibition of ROCK1 and ROCK2 affects dendritic spine morphology. Hippocampal neurons were transfected with plasmids expressing fluorescently labeled Lifeact, a small actin binding peptide, and then incubated with or without Y-27632, an established pan-ROCK small molecule inhibitor. Using an automated 3D spine morphometry analysis method, we showed that inhibition of ROCK1 and ROCK2 significantly increased the mean protrusion density and significantly reduced the mean protrusion width. A trending increase in mean protrusion length was observed following Y-27632 treatment, and novel effects were observed among spine classes. Exposure to Y-27632 significantly increased the number of filopodia and thin spines, while the numbers of stubby and mushroom spines were similar to mock-treated samples. These findings support the hypothesis that pharmacologic inhibition of ROCK1 and ROCK2 may convey therapeutic benefit for neurologic disorders that feature dendritic spine loss or aberrant structural plasticity.

Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI-anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis.

Transcription activator-like effector nucleases (TALENs) emerged as powerful tools for locus-specific genome engineering. Due to the ease of TALEN assembly, the key to streamlining TALEN-induced mutagenesis lies in identifying efficient TALEN pairs and optimizing TALEN mRNA injection concentrations to minimize the effort to screen for mutant offspring. Here we present a simple methodology to quantitatively assess bi-allelic TALEN cutting, as well as approaches that permit accurate measures of somatic and germline mutation rates in Drosophila melanogaster. We report that percent lethality from pilot injection of candidate TALEN mRNAs into Lig4 null embryos can be used to effectively gauge bi-allelic TALEN cutting efficiency and occurs in a dose-dependent manner. This timely Lig4-dependent embryonic survival assay also applies to CRISPR/Cas9-mediated targeting. Moreover, the somatic mutation rate of individual G0 flies can be rapidly quantitated using SURVEYOR nuclease and capillary electrophoresis, and germline transmission rate determined by scoring progeny of G0 outcrosses. Together, these optimized methods provide an effective step-wise guide for routine TALEN-mediated gene editing in the fly.

The mitochondria-associated membrane (MAM) is an endoplasmic reticulum (ER) domain that forms contacts with mitochondria and accommodates Ca²⁺ transfer between the two organelles. The GTPase Rab32 regulates this function of the MAM via determining the localization of the Ca²⁺ regulatory transmembrane protein calnexin to the MAM. Another function of the MAM is the regulation of mitochondrial dynamics mediated by GTPases such as dynamin-related protein 1 (Drp1). Consistent with the importance of the MAM for mitochondrial dynamics and the role of Rab32 in MAM enrichment, the inactivation of Rab32 leads to mitochondrial collapse around the nucleus. However, Rab32 and related Rabs also perform intracellular functions at locations other than the MAM including melanosomal trafficking, autophagosome formation and maturation, and retrograde trafficking to the trans-Golgi network (TGN). This plethora of functions raises questions concerning the original cellular role of Rab32 in the last common ancestor of animals and its possible role in the last eukaryotic common ancestor (LECA). Our results now shed light on this conundrum and identify a role in Drp1-mediated mitochondrial dynamics as one common denominator of this group of Rabs, which includes the paralogues Rab32A and Rab32B, as well as the more recently derived Rab29 and Rab38 proteins. Moreover, we provide evidence that this mitochondrial function is dictated by the extent of ER-association of Rab32 family proteins.

Membrane fusion in the endocytic pathway is mediated by a protein machinery consistent of Rab GTPases, tethering factors and SNAREs. In yeast, the endosomal CORVET and lysosomal HOPS tethering complexes share 4 of their 6 subunits. The 2 additional subunits in each complex - Vps3 and Vps8 for CORVET, and the homologous Vps39 and Vps41 for HOPS - bind directly to Rab5 and Rab7, respectively. In humans, all subunits for HOPS have been described. However, human CORVET remains poorly characterized and a homolog of Vps3 is still missing. Here we characterize 2 previously identified Vps39 isoforms, hVps39-1/hVam6/TLP and hVps39-2/TRAP1, in yeast and HEK293 cells. None of them can compensate the loss of the endogenous yeast Vps39, though the specific interaction of hVps39-1 with the virus-specific LT protein was reproduced. Both human Vps39 proteins show a cytosolic localization in yeast and mammalian cells. However, hVps39-2/TRAP1 strongly co-localizes with co-expressed Rab5 and interacts directly with Rab5-GTP in vitro. We conclude that hVps39-2/TRAP1 is an endosomal protein and an effector of Rab5, suggesting a role of the protein as a subunit of the putative human CORVET complex.