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dictyNews Volume 18 Number 02
Dicty News
Electronic Edition
Volume 18, number 2
January 26, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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Abstracts
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Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex
Christine Daunderer and Ralph Grf
Adolf-Butenandt-Institut/Zellbiologie, Universitt Mnchen, Germany
Ralph.Graef@lrz.uni-muenchen.de
Eur. J. Cell Biol., in press
Gamma-tubulin plays an essential role in microtubule nucleation and
organization and occurs, besides its centrosomal localization, in the
cytosol, where it forms soluble complexes with other proteins. We
investigated the size and composition of gamma-tubulin-complexes in
Dictyostelium, using a mutant cell line in which the endogenous copy of
the gamma-tubulin gene had been replaced by a tagged version of the gene.
Dictyostelium gamma-tubulin complexes were generally much smaller than the
large gamma-tubulin ring complexes found in higher organisms. The stability
of the small Dictyostelium gamma-tubulin complexes depended strongly on the
purification conditions, with a striking stabilization of the complexes
under high salt conditions. Furthermore, we cloned the Dictyostelium homolog
of Spc97 and an almost complete sequence of the Dictyostelium homolog of
Spc98, which are both components of gamma-tubulin complexes in other
organisms. Both proteins localize to the centrosome in Dictyostelium
throughout the cell cycle and are also present in a cytosolic pool. We
could show that the prevailing small complex present in Dictyostelium
consists of DdSpc98 and gamma-tubulin, whereas DdSpc97 does not associate.
Dictyostelium is thus the first organism investigated so far where the
three proteins do not interact stably in the cytosol.
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Visualization of Actin Dynamics During Macropinocytosis and Exocytosis
Eunkyung Lee and David A. Knecht
Department of Molecular and Cell Biology, University of Connecticut
Storrs, CT 06269
Traffic, in press
Macropinocytosis and exocytosis of post-lysosomes have been visualized
using an GFP probe that binds specifically to F-actin filaments. F-actin
association with macropinocytosis begins as a V-shaped infolding of the
membrane. Vesicle enlargement occurs through an inward movement of the
proximal point of the V as well as an outward protrusion at the tip of the
V to form an elongated invagination. The protrusion eventually closes at
its distal margin to become a vesicle and is moved centripetally while
recovering its circular shape. The vesicle loses its actin coat within one
minute after internalization. One hour later, post-lysosomal vesicles
became very weakly surrounded by actin while still cytoplasmic. Some of
these vesicles moved to the plasma membrane, docked, and then expelled their
contents. Slightly before the vesicle content began to disappear, an
increase in F-actin association with the vesicle was observed. This was
followed by rapid contraction of the vesicle and then disappearance of the
actin signal once the internal content was released. These results show
that dynamic changes in actin filament association with the vesicle membrane
accompany both endocytosis and exocytosis.
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Dynacortin is a novel actin bundling protein that localizes to dynamic actin
structures.
Douglas N. Robinson*, Stephani S. Ocon, Ronald S. Rock and James A. Spudich
Department of Biochemistry, Stanford University School of Medicine, Stanford,
CA 94305-5307
*Contact information: Department of Cell Biology, Johns Hopkins Medical
Institute, 725 N. Wolfe St., Baltimore, MD 21205, Ph: 410-502-2850
Email: Douglas.Robinson@jhu.edu
J. Biol. Chem., In press
Dynacortin is a novel protein that was discovered in a genetic suppressor
screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line
devoid of the cleavage furrow actin-bundling protein, cortexillin I. While
dynacortin is highly enriched in the cortex, particularly in cell-surface
protrusions, it is excluded from the cleavage furrow cortex during
cytokinesis. Here, we describe the biochemical characterization of this new
protein. Purified dynacortin is an 80-kDa dimer with a large 5.7 nm Stoke s
radius. Dynacortin cross-links actin filaments into parallel arrays with a
mole ratio of one dimer to 1.3 actin monomers and a 3.1 mM Kd. Using total
internal reflection fluorescence microscopy, GFP-dynacortin and the actin-
bundling protein coronin-GFP are seen to concentrate in highly dynamic
cortical structures with assembly and disassembly half-lives of about 15
seconds. These results indicate that cells have evolved different actin-
filament cross-linking proteins with complementary cellular distributions
that collaborate to orchestrate complex cell shape changes.
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Morphology and dynamics of the endocytic pathway in Dictyostelium discoideum
Eva M. Neuhaus, Wolfhard Almers and Thierry Soldati
Mol. Biol. Cell, in press
Dictyostelium discoideum is a genetically and biochemically tractable
social amoeba belonging to the crown group of eukaryotes. It performs some
of the tasks characteristic of a leukocyte such as chemotactic motility,
macropinocytosis and phagocytosis that are not performed by other model
organisms or are difficult to study. D. discoideum is becoming a popular
system to study molecular mechanisms of endocytosis, but the morphological
characterization of the organelles along this pathway and the comparison
with equivalent and/or different organelles in animal cells and yeasts were
lagging. Here, we used a combination of evanescent wave microscopy and
electron microscopy of rapidly frozen samples to visualize primary endocytic
vesicles, vesicular-tubular structures of the early and late endo-lysosomal
system, such as multi-vesicular bodies, and the specialized secretory
lysosomes. In addition, we present biochemical and morphological evidence
for the existence of a micropinocytic pathway, which contributes to the
uptake of membrane alongside macropinocytosis, which is the major fluid
phase uptake process. This complex endosomal compartment underwent
continuous cycles of tubulation/vesiculation as well as homo- and
heterotypic fusions, in a way very reminiscent of mechanisms and structures
documented in leukocytes. Finally, egestion of fluid phase from the
secretory lysosomes was directly observed.
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The dynamin A ring complex: molecular organisation and nucleotide-dependent
conformational changes
Boris Klockow, Willem Tichelaar*, Dean R. Madden*, Hartmut H. Niemann,
Toshihiko Akiba, Keiko Hirose$, Dietmar J. Manstein
Department of Biophysics and *Ion Channel Structure Research Group,
Max-Planck-Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg,
Germany National Institute for Advanced Interdisciplinary Research and
$ Gene Discovery Research Center, National Institute of Advanced Industrial
Science and Technology (AIST), 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan
The EMBO Journal, in press.
The GTPase dynamin A of the lower eukaryote Dictyostelium discoideum
functions in multiple membrane severing events. Here we show by electron
microscopy that dynamin A self-assembles into rings and helices similar to
human dynamin 1. Single particle analysis of the rings led to a three-
dimensional map of the nucleotide-free dynamin A ring complex. The complex
consists of two layers, each layer comprising two concentric rings with 11-
fold symmetry. The inner ring forms spike-like structures towards the centre,
ideally situated to penetrate into a tubulated lipid membrane. The GTP-bound
state, visualised using the non-hydrolysable GTP analogue GppNHp, is
associated with reorganisation of the ring complex leading to the generation
of short helical assemblies. Subsequent steps in the hydrolytic cycle induce
further conformational changes including an increase in helical pitch and
culminate in the disassembly of the complex. The results support a model for
the mechanochemical action of dynamin family proteins that involves both
constriction and stretching in membrane fission.
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[End Dicty News, volume 18, number 2]
