The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.

No abstract

EMMPRIN is a transmembrane glycoprotein expressed at high levels by tumor cells. It has been identified as a tumor-derived factor that can stimulate matrix metalloproteinase expression in fibroblasts and hence facilitate tumor invasion and metastasis. Recent studies have shown that full-length EMMPRIN is released by tumor cells, but the mechanism of release remains unclear. Here, we show that EMMPRIN is released from the surface of NCI-H460 cells via microvesicle shedding. However, these vesicles are unstable and rapidly break down to release bioactive EMMPRIN. Although microvesicle shedding has been considered a constitutive process in tumor cells, our data show that it can be amplified upon cell exposure to PMA, elucidating at least one signalling cascade responsible for EMMPRIN release. This pathway is dependent on protein kinase C, calcium mobilization and mitogen-activated protein kinase kinase (MEK 1/2). Thus, the results outline a novel form of tumor-stromal interaction in which extracellular matrix degradation by fibroblasts is controlled through the microvesicular release of EMMPRIN from tumor cells.

Herpes simplex virus type I (HSV) typically enters peripheral nerve terminals and then travels back along the nerve to reach the neuronal cell body, where it replicates or enters latency. To monitor axoplasmic transport of HSV, we used the giant axon of the squid, Loligo pealei, a well known system for the study of axoplasmic transport. To deliver HSV into the axoplasm, viral particles stripped of their envelopes by detergent were injected into the giant axon, thereby bypassing the infective process. Labeling the viral tegument protein, VP16, with green fluorescent protein allowed viral particles moving inside the axon to be imaged by confocal microscopy. Viral particles moved 2.2 ؎ 0.26 m͞sec in the retrograde direction, a rate comparable to that of the transport of endogenous organelles and of virus in mammalian neurons in culture. Electron microscopy confirmed that 96% of motile (stripped) viral particles had lost their envelope but retained tegument, and Western blot analysis revealed that these particles had retained protein from capsid but not envelope. We conclude that (i) HSV recruits the squid retrograde transport machinery; (ii) viral tegument and capsid but not envelope are sufficient for this recruitment; and (iii) the giant axon of the squid provides a unique system to dissect the viral components required for transport and to identify the cellular transport mechanisms they recruit.

We have assessed the effects of 15 pigmentation mutations on the development of retinal ganglion cell projections in mice in two ways: (1) by analyzing the pattern of innervation of the ipsilateral lateral geniculate nucleus as mapped in autoradiograms of brains of animals killed 12 days after intravitreal injection of 3H-proline into one eye and (2) by determining the ratio of axonally transported radioactive protein in the contralateral and ipsilateral optic tracts after similar intravitreal injections. Analysis of the ratio of transported protein in the two optic tracts provides a new and useful assay of the degree of decussation in experimental animals. The effects of the mutations on eye pigmentation, whole eye melanin content and relative tyrosinase activity also were examined. The degree of ipsilateral innervation generally correlates with the degree of pigmentation of the retinal pigment epithelium and with tyrosinase activity. However, discrepancies have been found in ch and ce mutants. In these animals the pigment epithelium is well pigmented, and the area of ipsilateral innervation in the lateral geniculate nucleus is extensive, despite a high ratio of label in contralateral to ipsilateral optic tracts and low tyrosinase activity. Furthermore, mice heterozygous for the c2J allele have pigmentation and optic projections that are normal even though tyrosinase is reduced to 40% of normal. The few anomalous results suggest that alternative or additional factors may control optic axon projections.