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First published online 20 May 2003
doi: 10.1242/jcs.00478

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Regulation of early endocytic vesicle motility and fission in a reconstituted system

¹ Marion Bessin Liver Research Center
² Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

View larger version (39K): [in a new window]   Fig. 1. Colocalization of early endocytic vesicles with Rab4 but not with Rab5. Representative studies are shown in which Texas-Red–ASOR and rhodamine-labeled MTs are visualized in panels A and D. Rab4 is immunolocalized in panel B, while Rab5 is immunolocalized in panel E. Merged images (C,F) reveal that the majority of MT-bound ligand-containing vesicles are associated with Rab4 (C). There is little colocalization of MT-bound ASOR-containing vesicles with Rab5 (F), although Rab5 is associated with many vesicles that do not contain ASOR.

View larger version (129K): [in a new window]   Fig. 2. Immunofluorescence localization of Rab4 in cultured rat hepatocytes. Overnight cultured rat hepatocytes grown on collagen-coated glass coverslips were incubated with Texas-Red-labeled ASOR for 20 minutes, treated with streptolysin O to permeabilize the plasma membrane, and stained with anti-Rab4 monoclonal antibody and Cy2-labeled anti-mouse antibody. Merged images of confocal sections and corresponding phase images of two representative cells are presented, revealing punctate areas of overlap (arrows) between endocytosed ASOR (red) and Rab4 (green). Bar, 10 µm.

View larger version (12K): [in a new window]   Fig. 3. Colocalization of MT-bound early endocytic vesicles with increasing concentrations of antibodies against Rab4 and Rab5. Monoclonal antibodies against Rab4 or Rab5 at increasing concentrations were perfused into a chamber containing MT-bound Texas-Red–ASOR-labeled vesicles, incubated for 6 minutes and visualized after addition of Cy2-labeled secondary antibody. The plot indicates percent colocalization between MT-bound Texas-Red–ASOR vesicles with Rab4 and Rab5 antibody at the indicated concentrations.

View larger version (13K): [in a new window]   Fig. 4. The determination of affinities of anti-Rab4 and anti-Rab5 to GST-Rab fusion proteins by surface plasmon resonance. Surface plasmon resonance technology was used to measure the binding of anti-Rab4 monoclonal antibody to a cuvette coated with GST-Rab4 (A), and the binding of anti-Rab5 monoclonal antibody to a GST-Rab5-coated cuvette (B). Antibody was added at the indicated concentrations, binding was monitored for 10 minutes, cuvettes were acid washed to remove antibody, and antibody was re-added. An equilibrium plot of final signal versus antibody concentration (C) was used to fit a curve for the apparent affinities of the antibodies, yielding Kd values of 2 and 22 nM for anti-Rab4 and anti-Rab5 monoclonal antibodies, respectively. (D) Endocytic vesicles (total protein 60 µg) were incubated with 0.5 µM GST-Rab4 for 15 minutes at 37°C. Following extensive washing vesicles were immunoblotted with monoclonal antibody against Rab4.

View larger version (41K): [in a new window]   Fig. 5. Direct demonstration that Rab-GDI or monoclonal antibody to Rab4 release Rab4-GDP from early endocytic vesicles. Endocytic vesicles were preincubated with buffer or buffer plus 4 mM GDP or 4 mM GTP--S for 15 minutes at 37°C and subsequently incubated in the absence (A) or presence of Rab-GDI (B) or Rab4 monoclonal antibody (Rab4 mAb, C). After centrifugation, Rab4 was detected in supernatants or pellets by western blot as indicated. Rab-GDI caused Rab4 to appear in the supernatants and the amount of Rab4 was increased by preincubation of the vesicles with GDP and decreased by preincubation of the vesicles with GTP--S. Similarly, Rab4 mAb caused Rab4 to appear in the supernatants and the amount was increased by preincubation of the vesicles with GDP. The IgG band at 25 kDa results from crossreactivity of the anti-mouse secondary antibody with the Rab4 mAb that was incubated with the vesicles in the experiment.

View larger version (37K): [in a new window]   Fig. 6. The effect of guanine nucleotides on Rab4 immunolocalization on MT-bound endocytic vesicles. MT-bound Texas-Red–ASOR-containing early endocytic vesicles were incubated for 5 minutes in a motility chamber with buffer containing 4 mM GDP (top panels), or 4 mM GDP followed by an additional 5 minute incubation with 4 mM GTP--S (bottom panels). Monoclonal antibody against Rab4 was perfused into the chamber, incubated for 6 minutes, and visualized after addition of Cy2-labeled secondary antibody. Representative studies are shown in which Texas-Red–ASOR and rhodamine-labeled MTs are visualized in panels A and D. Rab 4 is immunolocalized in panels B and E. Merged images reveal that Rab4 is no longer immunolocalized to vesicles when pre-incubated with GDP (C). However, when GTP--S is perfused after GDP, Rab4 remains associated with these vesicles (F). These results are consistent with removal of Rab4-GDP but not Rab4-GTP from vesicles by the monoclonal antibody.

View larger version (87K): [in a new window]   Fig. 7. Pre-incubation of MT-bound vesicles with GDP results in increased motility and fission. Fluorescent early endocytic vesicles were prepared after injection of a rat with Texas-Red-labeled ASOR. Endocytic vesicles were flowed into a 3 µl microscopy chamber in which Taxol-stabilized rhodamine-labeled MTs had been attached to the glass surface. MT-bound vesicles were incubated for 5 minutes with buffer alone (A) or with buffer containing 4 mM GDP (B). Time in seconds after addition of 50 µM ATP is shown at the bottom of each panel. Arrows point to examples of vesicles bound to MTs. In subsequent panels, smaller arrows indicate motile vesicles while larger arrows point to the original vesicle position (A,B). In B, arrowheads point to vesicles undergoing fission. Bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 8. Effects of guanosine nucleotide analogues on MT-based motility and fission of early endocytic vesicles. MT-bound vesicles were incubated for 5 minutes with buffer alone (control) or with buffer containing 4 mM GTP--S, GDP--S, GDP, GDP plus 0.5 µM Rab-GDI, or 2 µM GST-Rab4 as indicated. Guanine nucleotides were exchanged into GST-Rab4 by preincubation immediately before addition to vesicles. ATP (50 µM) was added to produce vesicle motility, and this activity was scored. The bars in panel A indicate the percentage of MT-bound vesicles that moved upon ATP addition. For each experiment, the number of motile vesicles versus the total number of MT-bound vesicles that were examined is in parentheses. The bars in panel B indicate the percentage of moving vesicles that underwent fission. For each experiment, the number of vesicles that underwent fission versus the number of motile vesicles is in parentheses. *P<0.001 versus control.

View larger version (15K): [in a new window]   Fig. 9. GDP enhances minus-end-directed motility of early endocytic vesicles. Fluorescent-polarity-marked microtubules were prepared as described (Murray et al., 2000). MT-bound vesicles were incubated for 5 minutes with buffer alone or with buffer containing 4 mM GDP, following which 50 µM ATP was added to produce vesicle motility. The bars indicate the percentage of vesicles that moved upon ATP addition in the minus-end or plus-end directions. For each experiment, the number of motile vesicles in either direction versus the total number of motile vesicles that were examined is in parentheses. *P<0.01.

View larger version (24K): [in a new window]   Fig. 10. KIFC2 is highly enriched and functional on endocytic vesicles. (A) Fifteen micrograms of total protein from rat liver homogenate, PNS, cytosol, S-200 fraction and purified endocytic vesicles were subjected to 10-20% gradient SDS-Page and immunoblotted with antibody to KIFC2, as described in Materials and Methods. (B,C) MT-bound endocytic vesicles were incubated for 6 minutes with buffer alone or buffer containing affinity purified anti-KIFC2, following which 50 µM ATP was added to produce vesicle motility. In some studies, fluorescent polarity marked microtubules were used (C). The bars in panel B indicate the percentage of MT-bound vesicles that moved upon ATP addition. The number of motile vesicles versus the total number of MT-bound vesicles that were examined is in parentheses. The bars in panel C indicate the percentage of vesicles that moved upon ATP addition in the minus-end or plus-end directions. For each experiment, the number of motile vesicles in either direction versus the total number of motile vesicles that were examined is in parentheses. *P<0.002 versus control.

View larger version (33K): [in a new window]   Fig. 11. Model of the proposed mechanism for regulation by Rab4 of early endocytic vesicle sorting of the asialoglycoprotein receptor and ligand. The shaded area represents the cell exterior and the vesicle interior, the red tube represents a microtubule with its plus (+) and minus (-) ends specified, the arrow represents passage of time on the scale of tens of seconds. Ligand (ASOR) binds to its receptor (ASGPR) at the cell surface, where it is internalized into an endocytic vesicle. The vesicle binds to microtubules through plus- and minus-end-directed kinesins. GTP-Rab4 present on the vesicle inhibits minus-end kinesins (KIFC2). Hydrolysis of GTP on Rab4 releases this inhibition, allowing minus-end-directed movement of the vesicle, resulting in fission and sorting of its contents along the microtubule. For simplicity Rab4 is shown binding directly to the minus-end kinesin, and only the minus-end motor is shown moving. Endogenous Rab-GDI could remove Rab4-GDP from the vesicle, allowing Rab4 to be recycled. In cultured rat hepatocytes, ligand is eventually (after 30-60 minutes) sorted to lysosomes (Wolkoff et al., 1984), which reside near the cell center at the minus ends of microtubules. However, early endocytic sorting events do not demonstrate simple progression toward the cell center and instead show multiple plus- and minus-end-directed movements (Murray et al., 2000).