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Microtubule and motor-dependent endocytic vesicle sorting in vitro.

Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW - J. Cell Biol. (2000)

Bottom Line: After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation.Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles.Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies.

View Article: PubMed Central - PubMed

Affiliation: Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

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Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.
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Figure 6: Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.

Mentions: There is a possibility that proteins in the motor fraction that were used to coat the glass to which MTs were attached could bind substantial amounts of vanadate, thus falsely suggesting that it is without effect in the endosomal motility and fission assays. For this reason, a series of experiments was performed using MTs that were bound to glass via DEAE-dextran (Fig. 6). In addition, studies were performed at ATP concentrations of either 50 μM or 4 mM, in the absence of a regenerating system. As seen in Fig. 6, vanadate at concentrations as high as 20 μM in the presence of 4 mM ATP had no effect on vesicle motility or fission. Under these conditions, vanadate should preferentially inhibit dynein function. At concentrations of 50 μM or above, motility was reduced. Although the fraction of fission events in motile vesicles remained unchanged (Fig. 6 right), the absolute number of fissions was reduced in proportion to the reduction in motility. At these high concentrations, vanadate is known to inhibit many ATPases, including kinesins (Brady 1991; Vale et al. 1992). Vesicle motility was markedly reduced by 1 mM AMP-PNP even at high (4 mM) concentrations of ATP, and no fission events were seen. Under these conditions, AMP-PNP should preferentially inhibit function of kinesins.


Microtubule and motor-dependent endocytic vesicle sorting in vitro.

Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW - J. Cell Biol. (2000)

Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2189808&req=5

Figure 6: Effects of vanadate and AMP-PNP on vesicle motility and fission. MTs were bound to the glass chamber via DEAE-dextran. Studies were performed at ATP concentrations of 50 μM (black bars) or 4 mM (cross-hatched bars) in the absence of a regenerating system. The bars in the left graph indicate the percentage of MT-bound vesicles that moved upon ATP addition in the presence or absence of varied concentrations of vanadate or 1 mM AMP-PNP. The total number of MT-bound vesicles that were examined in each experiment is in parentheses. The bars in the right graph indicate the percentage of moving vesicles that underwent fission.
Mentions: There is a possibility that proteins in the motor fraction that were used to coat the glass to which MTs were attached could bind substantial amounts of vanadate, thus falsely suggesting that it is without effect in the endosomal motility and fission assays. For this reason, a series of experiments was performed using MTs that were bound to glass via DEAE-dextran (Fig. 6). In addition, studies were performed at ATP concentrations of either 50 μM or 4 mM, in the absence of a regenerating system. As seen in Fig. 6, vanadate at concentrations as high as 20 μM in the presence of 4 mM ATP had no effect on vesicle motility or fission. Under these conditions, vanadate should preferentially inhibit dynein function. At concentrations of 50 μM or above, motility was reduced. Although the fraction of fission events in motile vesicles remained unchanged (Fig. 6 right), the absolute number of fissions was reduced in proportion to the reduction in motility. At these high concentrations, vanadate is known to inhibit many ATPases, including kinesins (Brady 1991; Vale et al. 1992). Vesicle motility was markedly reduced by 1 mM AMP-PNP even at high (4 mM) concentrations of ATP, and no fission events were seen. Under these conditions, AMP-PNP should preferentially inhibit function of kinesins.

Bottom Line: After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation.Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles.Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies.

View Article: PubMed Central - PubMed

Affiliation: Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Show MeSH
Related in: MedlinePlus