``Frustrated Exocytosis'' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

``Frustrated Exocytosis'' — A Novel Phenomenon: Membrane Fusion without Contents Release,... The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca2+] i transient and exocytosis of dense core vesicles (``trichocysts'') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca2+ ([Ca2+] o = 50 μm). When [Ca2+] o is kept at 30 nm (<[Ca2+]rest i ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca2+ o . Upon readdition of Ca2+ o or some other appropriate Me2+ o at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca2+, Sr2+ or Mn2+ are vesicular, but when formed in presence of Mg2+, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg2+] o = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis'') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg2+] o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me2+] to maintain docking sites in a functional state, (ii) requirement of Ca2+ o or of some other Me2+ o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty'' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled'' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis''. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

``Frustrated Exocytosis'' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

Loading next page...
 
/lp/springer_journal/frustrated-exocytosis-a-novel-phenomenon-membrane-fusion-without-4Ge6sVciYl
Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 2000 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232001093
Publisher site
See Article on Publisher Site

Abstract

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca2+] i transient and exocytosis of dense core vesicles (``trichocysts'') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca2+ ([Ca2+] o = 50 μm). When [Ca2+] o is kept at 30 nm (<[Ca2+]rest i ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca2+ o . Upon readdition of Ca2+ o or some other appropriate Me2+ o at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca2+, Sr2+ or Mn2+ are vesicular, but when formed in presence of Mg2+, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg2+] o = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis'') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg2+] o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me2+] to maintain docking sites in a functional state, (ii) requirement of Ca2+ o or of some other Me2+ o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty'' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled'' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis''.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Aug 1, 2000

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off