Transmission of HIV-1 via intercellular connections has been estimated as 100-1000 times more efficient than a cell-free process, perhaps in part explaining persistent viral spread in the presence of neutralizing antibodies. Such effective intercellular transfer of HIV-1 could occur through virological synapses or target-cell filopodia connected to infected cells. Here we report that membrane nanotubes, formed when T cells make contact and subsequently part, provide a new route for HIV-1 transmission. Membrane nanotubes are known to connect various cell types, including neuronal and immune cells, and allow calcium-mediated signals to spread between connected myeloid cells. However, T-cell nanotubes are distinct from open-ended membranous tethers between other cell types, as a dynamic junction persists within T-cell nanotubes or at their contact with cell bodies. We also report that an extracellular matrix scaffold allows T-cell nanotubes to adopt variably shaped contours. HIV-1 transfers to uninfected T cells through nanotubes in a receptor-dependent manner. These data lead us to propose that HIV-1 can spread using nanotubular connections formed by short-term intercellular unions in which T cells specialize.

hours after transfection. COS-7 cells were incubated with EGF (0.1 g/ml) [biotinylated, complexed to Texas Red-streptoavidin (Molecular Probes, Eugene, OR)] in binding buffer [20 mM Hepes-NaOH ( pH 7.5), 130 mM NaCl, and 0.1% bovine serum albumin] at 4°C for 60 min. Internalization of EGF was allowed by incubation in Dulbecco's modified Eagle's medium at 37°C for 10 min, then excess EGF was removed with 0.2 M AcOH ( pH 2.5) and 0.5 M NaCl at 4°C for 5 min. Cells were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with a polyclonal antibody to myc (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein isothiocyanate-conjugated antibody to rabbit (Organon Teknika, Boxtel, Netherlands). Internalization of EGF was observed by confocal microscopy (Bio-Rad). 37. We thank Y. Watanabe (Ehime University, Japan) for providing us with various synthetic phosphoinositides.

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Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using ~2h-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 1251 and gold colloid. Using 1251-transferrin we have shown that, whereas at 37°C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37°C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 1251-ATR displayed the same kinetics of uptake but following its internalization at 37°C, it was partially degraded. At 22°C and below, the intracellular degradation of ~251-ATR was selectively inhibited and as a result it accumulated intracellularly.Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22°C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22°C, no other compartments became labeled but if they were warmed to 37°C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22°C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

Abstract. We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRPloaded lysosomes are lysosomal-associated membrane protein-1 (LAMP-l) positive, mannose-6-phosphate receptor (M6PR) negative, and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded.Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact.Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step.

Rab GTPases are regulators of intracellular membrane traffic. We report a possible function of Rab27a, a protein implicated in several diseases, including Griscelli syndrome, choroideremia, and the Hermansky-Pudlak syndrome mouse model, gunmetal. We studied endogenous Rab27a and overexpressed enhanced GFP-Rab27a fusion protein in several cultured melanocyte and melanoma-derived cell lines. In pigmented cells, we observed that Rab27a decorates melanosomes, whereas in nonpigmented cells Rab27a colocalizes with melanosome-resident proteins. When dominant interfering Rab27a mutants were expressed in pigmented cells, we observed a redistribution of pigment granules with perinuclear clustering. This phenotype is similar to that observed by others in melanocytes derived from the ashen and dilute mutant mice, which bear mutations in the Rab27a and MyoVa loci, respectively. We also found that myosinVa coimmunoprecipitates with Rab27a in extracts from melanocytes and that both Rab27a and myosinVa colocalize on the cytoplasmic face of peripheral melanosomes in wild-type melanocytes. However, the amount of myosinVa in melanosomes from Rab27a-deficient ashen melanocytes is greatly reduced. These results, together with recent data implicating myosinVa in the peripheral capture of melanosomes, suggest that Rab27a is necessary for the recruitment of myosinVa, so allowing the peripheral retention of melanosomes in melanocytes.

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