Microtubule-dependent movement of late endocytic vesicles in vitro

Requirements for dynein and kinesin

Eustratios Bananis, Sangeeta Nath, Kristie Gordon, Peter Satir, Richard J. Stockert, John W. Murray, Allan W. Wolkoff

Research output: Contribution to journalArticle

85 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)3688-3697
Number of pages10
JournalMolecular Biology of the Cell
Volume15
Issue number8
DOIs
StatePublished - Aug 2004

Fingerprint

Dyneins
Kinesin
Transport Vesicles
Microtubules
Antibodies
Asialoglycoprotein Receptor
Proteomics
Microscopy
Flow Cytometry
Proteins
In Vitro Techniques
Injections
Liver

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Microtubule-dependent movement of late endocytic vesicles in vitro : Requirements for dynein and kinesin. / Bananis, Eustratios; Nath, Sangeeta; Gordon, Kristie; Satir, Peter; Stockert, Richard J.; Murray, John W.; Wolkoff, Allan W.

In: Molecular Biology of the Cell, Vol. 15, No. 8, 08.2004, p. 3688-3697.

Research output: Contribution to journalArticle

Bananis, Eustratios ; Nath, Sangeeta ; Gordon, Kristie ; Satir, Peter ; Stockert, Richard J. ; Murray, John W. ; Wolkoff, Allan W. / Microtubule-dependent movement of late endocytic vesicles in vitro : Requirements for dynein and kinesin. In: Molecular Biology of the Cell. 2004 ; Vol. 15, No. 8. pp. 3688-3697.
@article{525d617d9d9843f5aecb7fd3c601c39e,
title = "Microtubule-dependent movement of late endocytic vesicles in vitro: Requirements for dynein and kinesin",
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.",
author = "Eustratios Bananis and Sangeeta Nath and Kristie Gordon and Peter Satir and Stockert, {Richard J.} and Murray, {John W.} and Wolkoff, {Allan W.}",
year = "2004",
month = "8",
doi = "10.1091/mbc.E04-04-0278",
language = "English (US)",
volume = "15",
pages = "3688--3697",
journal = "Molecular Biology of the Cell",
issn = "1059-1524",
publisher = "American Society for Cell Biology",
number = "8",

}

TY - JOUR

T1 - Microtubule-dependent movement of late endocytic vesicles in vitro

T2 - Requirements for dynein and kinesin

AU - Bananis, Eustratios

AU - Nath, Sangeeta

AU - Gordon, Kristie

AU - Satir, Peter

AU - Stockert, Richard J.

AU - Murray, John W.

AU - Wolkoff, Allan W.

PY - 2004/8

Y1 - 2004/8

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=3343021872&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3343021872&partnerID=8YFLogxK

U2 - 10.1091/mbc.E04-04-0278

DO - 10.1091/mbc.E04-04-0278

M3 - Article

VL - 15

SP - 3688

EP - 3697

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 8

ER -