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, 15 (8), 3688-97

Microtubule-dependent Movement of Late Endocytic Vesicles in Vitro: Requirements for Dynein and Kinesin

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Microtubule-dependent Movement of Late Endocytic Vesicles in Vitro: Requirements for Dynein and Kinesin

Eustratios Bananis et al. Mol Biol Cell.

Abstract

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.

Figures

Figure 1.
Figure 1.
Schematic diagram of the flow cytometric purification of fluorescent endocytic vesicles. Presorted vesicles (a) were assessed from the dot plot representation of forward scatter (FSC) and pulse width (I) and set on a linear scale. Approximately, 90–95% of these vesicles (c) were gated and assessed for forward (FSC) and side scatter (SSC; II) set on a linear scale. From this, ∼95% of vesicles (f) were gated for fluorescence analysis (III).
Figure 2.
Figure 2.
Immunofluorescence detection of endocytic vesicle-associated proteins. Texas-red–labeled ASOR-containing early (left panels) or late (right panels) endocytic vesicles were bound to rhodamine-labeled taxol-stabilized MTs. MT-bound vesicles were incubated for 6 min with primary antibodies against the indicated proteins, followed by addition of appropriate Cy2-labeled secondary antibodies (green). Corresponding images from both channels were merged. Colocalization between vesicles and proteins results in yellow vesicles. These studies indicate that early vesicles associate with ASGPR (receptor), conventional kinesin I (KIF5B, kinesin I [HC]), Rab4 and KIFC2, whereas late vesicles associate with KIFC2, KIF3A (kinesin II), Rab7, dynein, and dynactin (p150). MT-bound vesicles that do not contain fluorescent ligand are also seen associated with these proteins. HC, heavy chain; IC, intermediate chain.
Figure 3.
Figure 3.
Quantitation of endocytic vesicle associated proteins as determined by immunofluorescence. Quantitation of colocalization studies as in Figure 2, was performed as described in MATERIALS AND METHODS. The total number of MT-bound early and late vesicles examined is shown in parentheses. Asterisk indicates published data (Bananis et al., 2003).
Figure 4.
Figure 4.
Microtubule-based motility and fission of early and late endocytic vesicles. Texas-red–labeled early and late endocytic vesicles were bound to rhodamine-labeled microtubules (MTs) within a glass microscopy chamber. Motility (A) and fission (B) of these vesicles were quantified following addition of 50 μM ATP. The total number of MT-bound vesicles and the total number of motile vesicles that were examined are shown in parentheses in A and B, respectively.
Figure 5.
Figure 5.
Effects of vanadate and AMP-PNP on motility of late endocytic vesicles. Late endocytic vesicles were perfused into the chamber and bound to MTs. The bars indicate the percentage of vesicles that moved along MTs following addition of 50 μM ATP in control buffer or in the presence of 5 μM vanadate or 1 mM AMP-PNP. The total number of vesicles examined is shown in parentheses. *p < 0.0001 compared with buffer alone.
Figure 6.
Figure 6.
Directional motility of early and late endocytic vesicles. Polarity-marked MTs were prepared and bound to the inner surface of a glass microscopy chamber. Early or late endocytic vesicles were then perfused into the chamber and bound to MTs. The bars indicate the percentage of early (left) and late (right) endocytic vesicles that moved toward the minus-end of MTs after addition of 50 μM ATP in the presence or absence of 5 μM vanadate. The total number of motile vesicles examined is shown in parentheses. *p < 0.002 compared with control late vesicles.
Figure 7.
Figure 7.
Effect of specific motor antibodies on late endocytic vesicle motility. MT-bound late endocytic vesicles were incubated with antibodies to specific motor proteins, as indicated, for 6 min. Motility was then quantified after 50 μM ATP addition. The total number of MT-bound vesicles examined is shown in parentheses. *p < 0.0004 compared with buffer or nonimmune IgG.
Figure 8.
Figure 8.
Effect of preincubation with dynamitin antibody on attachment of endocytic vesicles to MTs. Endocytic vesicles were preincubated with mAb to dynamitin (p50 subunit of dynactin complex) or nonimmune mouse IgG (control) for 6 min. After incubation, vesicles were perfused into MT-coated glass microscopy chambers. (A) The bars indicate the number of fluorescent vesicles that bound per μm microtubule length. Results shown are mean and SD. (B) Immunofluorescence localization of the indicated proteins on MT-attached late endocytic vesicles with or without preincubation with antidynamitin antibody. The total number of MT-bound vesicles examined for each experiment is shown in parentheses. *p < 0.0001 compared with each control.
Figure 9.
Figure 9.
Flow cytometric analysis and purification of fluorescent endocytic vesicles. Alexa 488–labeled endocytic vesicles were prepared and sorted on a Dykocytomation (MoFLo) Cell Sorter. Regions indicated by lower case letters in circles in this figure correspond to those indicated schematically in Figure 1. (A) Dot plot representation of forward scatter (FSC) vs. pulse width of presorted vesicles. (B) Dot plot representation of FSC vs. side scatter (SSC) of vesicles within region (c) of dot plot (B). (C) and (D) Fluorescent profiles of unlabeled (control) or early (C) or late (D) endocytic vesicles within region (f) of dot plot (B).
Figure 10.
Figure 10.
Flow cytometric analysis of purified vesicles colabeled with DiD. Presorted vesicles were incubated with DiD, a fluorescent general membrane marker. (A) Mixed fluorescent and nonfluorescent vesicles from region (f) in Figure 9B were analyzed for Alexa 488 and DiD fluorescence. The boxed area depicts vesicles that have both fluorescent membrane dye and fluorescent ASOR and represents 60% of total vesicles. (B) The Alexa 488 fluorescence profile of the total population of vesicles from A is shown. (C) Sorted fluorescent vesicles from region (h) in Figure 9D were analyzed for Alexa 488 and DiD fluorescence. Ninety-nine percent of total vesicles were within the boxed area. (D) The Alexa 488 fluorescence profile of the total population of vesicles from C is shown.
Figure 11.
Figure 11.
Immunoblot analysis of early and late endocytic vesicle populations after flow cytometric purification. Immunoblots were performed after 10–20% gradient SDS-PAGE (8 μg of protein per lane). (A) Presorted vesicles (Figure 1a) and sorted fluorescent early and late endocytic vesicles (Figure 1h) were examined for the presence of the indicated proteins. Results of a representative experiment are shown. (B) Presorted (Figure 1a), fluorescent (late) (Figure 1h), unlabeled (Figure 1g), small (Figure 1d) and aggregated (Figure 1b) late endocytic vesicles were subjected to immunoblot analysis for the ASGPR, Rab4, and Rab5 as indicated.

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