Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein

Irene Wacker, Christoph Kaether, Andreas Krömer, Andrea Migala, Wolfhard Almers, Hans Hermann Gerdes

Research output: Contribution to journalArticle

185 Citations (Scopus)

Abstract

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Videomicroscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Original languageEnglish (US)
Pages (from-to)1453-1463
Number of pages11
JournalJournal of Cell Science
Volume110
Issue number13
StatePublished - 1997
Externally publishedYes

Fingerprint

Transport Vesicles
Secretory Vesicles
Green Fluorescent Proteins
Microtubules
trans-Golgi Network
Proteins
Cell Membrane
Nocodazole
Fluorometry
Cathepsin D
Vero Cells
Butyrates
Protein Transport
Transferrin
Recombinant Proteins
Clone Cells
Observation
human CHGB protein
Membranes

Keywords

  • Chromogranin B
  • GFP
  • Microtubule
  • Real time
  • Secretory vesicle
  • TGN

ASJC Scopus subject areas

  • Cell Biology

Cite this

Wacker, I., Kaether, C., Krömer, A., Migala, A., Almers, W., & Gerdes, H. H. (1997). Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. Journal of Cell Science, 110(13), 1453-1463.

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. / Wacker, Irene; Kaether, Christoph; Krömer, Andreas; Migala, Andrea; Almers, Wolfhard; Gerdes, Hans Hermann.

In: Journal of Cell Science, Vol. 110, No. 13, 1997, p. 1453-1463.

Research output: Contribution to journalArticle

Wacker, I, Kaether, C, Krömer, A, Migala, A, Almers, W & Gerdes, HH 1997, 'Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein', Journal of Cell Science, vol. 110, no. 13, pp. 1453-1463.
Wacker, Irene ; Kaether, Christoph ; Krömer, Andreas ; Migala, Andrea ; Almers, Wolfhard ; Gerdes, Hans Hermann. / Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein. In: Journal of Cell Science. 1997 ; Vol. 110, No. 13. pp. 1453-1463.
@article{86e20572d6a64e27add939391921803d,
title = "Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein",
abstract = "Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Videomicroscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50{\%} of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.",
keywords = "Chromogranin B, GFP, Microtubule, Real time, Secretory vesicle, TGN",
author = "Irene Wacker and Christoph Kaether and Andreas Kr{\"o}mer and Andrea Migala and Wolfhard Almers and Gerdes, {Hans Hermann}",
year = "1997",
language = "English (US)",
volume = "110",
pages = "1453--1463",
journal = "Journal of Cell Science",
issn = "0021-9533",
publisher = "Company of Biologists Ltd",
number = "13",

}

TY - JOUR

T1 - Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein

AU - Wacker, Irene

AU - Kaether, Christoph

AU - Krömer, Andreas

AU - Migala, Andrea

AU - Almers, Wolfhard

AU - Gerdes, Hans Hermann

PY - 1997

Y1 - 1997

N2 - Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Videomicroscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

AB - Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20°C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37°C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Videomicroscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 μm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

KW - Chromogranin B

KW - GFP

KW - Microtubule

KW - Real time

KW - Secretory vesicle

KW - TGN

UR - http://www.scopus.com/inward/record.url?scp=0030747463&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030747463&partnerID=8YFLogxK

M3 - Article

VL - 110

SP - 1453

EP - 1463

JO - Journal of Cell Science

JF - Journal of Cell Science

SN - 0021-9533

IS - 13

ER -