Abstract. We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (cMoroquine or NH4CI). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgpL20, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (",,90%) within •20 min, indicating that as the intraendosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NI-LCl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that (a) the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, (b) ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and (c) endosomes rather than secondary lysos0mes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysoSomal enzymes meet. The implications of these findings for the biogenesis of secondary lysosomes are discussed. M ANNOSE-6-PHOSPHATE (Man6P) 1 receptors (215 kD) are known to function in the targeting and transport of newly synthesized lysosomal enzymes (for reviews see references 11 and 43). To identify the transport pathway followed by the 215-kD Man6P receptor, we previously localized these receptors in a variety of cell types by immunocytochemistry (4, 5, 7) and showed that they are concentrated in the Golgi complex, in small vesicles (often with clathrin-like coats), and in larger vacuoles. These organelles were assumed to represent the sorting site, the carrier, and the delivery site, respectively, for lysosomal enzymes. We also reported that treatment of cells for 3 h with lysosomotropic weak bases (chloroquine or NILCI) causes depletion of Man6P receptors from the sorting site in the Golgi complex and their accumulation ...

The human CMV UL37x1-encoded protein, also known as the viral mitochondria-localized inhibitor of apoptosis, traffics to the endoplasmic reticulum and mitochondria of infected cells. It induces the fragmentation of mitochondria and blocks apoptosis. We demonstrate that UL37x1 protein mobilizes Ca 2؉ from the endoplasmic reticulum into the cytosol. This release is accompanied by cell rounding, cell swelling, and reorganization of the actin cytoskeleton, and these morphological changes can be substantially blocked by a Ca 2؉ chelating agent. The UL37x1-mediated release of Ca 2؉ from the endoplasmic reticulum likely has multiple consequences, including induction of the unfolded protein response, modulation of mitochondrial function, induction of mitochondrial fission, and protection against apoptotic stimuli.actin cytoskeleton ͉ apoptosis ͉ cytopathic effect ͉ mitochondrial fission ͉ viral mitochondria-localized inhibitor of apoptosis

Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.

During Drosophila oogenesis, the targeted localization of gurken (grk) mRNA leads to the establishment of the axis polarity of the egg. In early stages of oogenesis, grk mRNA is found at the posterior of the oocyte, whereas in the later stages grk mRNA is positioned at the dorsal anterior corner of the oocyte. In order to visualize the real-time localization and anchorage of endogenous grk mRNA in living oocytes, we have utilized the MS2-MCP system. We show that MCP-GFP-tagged endogenous grk mRNA localizes properly within wild-type oocytes and behaves aberrantly in mutant backgrounds. Fluorescence recovery after photobleaching (FRAP) experiments of localized grk mRNA in egg chambers reveal a difference in the dynamics of grk mRNA between young and older egg chambers. grk mRNA particles, as a population, are highly dynamic molecules that steadily lose their dynamic nature as oogenesis progresses. This difference in dynamics is attenuated in K10 and sqd1 mutants such that mislocalized grk mRNA in older stages is much more dynamic compared with that in wild-type controls. By contrast, in flies with compromised dynein activity, properly localized grk mRNA is much more static. Taken together, we have observed the nature of localized grk mRNA in live oocytes and propose that its maintenance changes from a dynamic to a static process as oogenesis progresses.

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