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dictyNews Volume 27 Number 05
dictyNews
Electronic Edition
Volume 27, number 5
August 18, 2006
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Selective membrane exclusion in phagocytic and macropinocytic cups
Valentina Mercanti1#, Steve J. Charette1#, Nelly Bennett2, Jean-Jeacques
Ryckewaert3, Franois Letourneur4 and Pierre Cosson1
1. Universite de Geneve, Centre Medical Universitaire, Departement de
Physiologie Cellulaire et Metabolisme, 1 rue Michel Servet,
CH-1211 Geneve 4, Switzerland.
2. Laboratoire de Biochimie et Biophysique des Systemes Integres,
Departement de Reponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des
Martyrs, 38054 Grenoble Cedex 9, France.
3. Laboratoire de Chemie des Proteines, ERM 201 INSERM/CEA/UJF, 17 rue des
Martyrs, 38054 Grenoble Cedex 9, France.
4. Institut de Biologie et Chimie des Proteines (IBCP UMR 5086); CNRS;
Univ. Lyon1; IFR128 BioSciences Lyon-Gerland; 7, passage du Vercors,
69367 Lyon cedex 07, France
#These authors contributed equally to this work.
Journal of Cell Science, in press
Specialized eukaryotic cells can ingest large particles and sequester them
within membrane-delimited phagosomes. Many studies have described the
delivery of lysosomal proteins to the phagosome, but little is known about
membrane sorting during the early stages of phagosome formation. Here we
used Dictyostelium discoideum amoebae to analyze the membrane composition
of newly formed phagosomes. The membrane delimiting the closing phagocytic
cup was essentially derived from the plasma membrane, but a subgroup of
proteins was specifically excluded. Interestingly the same phenomenon was
observed during the formation of macropinosomes, suggesting that the same
sorting mechanisms are at play during phagocytosis and macropinocytosis.
Analysis of mutant strains revealed that clathrin-associated adaptor
complexes AP-1, -2 and -3 were not necessary for this selective exclusion,
and accordingly ultrastructural analysis revealed no evidence for vesicular
transport around phagocytic cups. Our results suggest the existence of a
new, as yet uncharacterized, sorting mechanism in phagocytic and
macropinocytic cups.
Submitted by: Steve Charette [steve.charette@medecine.unige.ch]
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The function of the Dictyostelium Atg1 kinase during autophagy and
development
Turgay Tekinay, Mary Y. Wu1, Grant P. Otto, O. Roger Anderson and Richard H.
Kessin
Eukaryotic Cell, in press
When starved, the amoebae of Dictyostelium discoideum initiate a
developmental process that results in the formation of fruiting bodies, in
which stalks support balls of spores. The nutrients and energy necessary for
development are provided by autophagy. Atg1 is a protein kinase that
regulates the induction of autophagy in budding yeast. In addition to a
conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has
significant homology to the Caenorhabditis elegans and mammalian Atg1
homologues, but not to the budding yeast Atg1. We investigated the function
of the kinase and conserved C-terminal domains of DdAtg1 and showed that
these domains are essential for autophagy and development. Kinase-negative
DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype
when expressed in the wild-type cells. GFP-tagged kinase-negative DdAtg1
co-localizes with RFP tagged DdAtg8, a marker of preautophagosomal structures
and autophagosomes. The conserved C-terminal region is essential for
localization of kinase-negative DdAtg1 to autophagosomes labeled with
RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the
kinase-defective mutant also depends on the C-terminal domain. In cells
expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate
but seem not to be functional. By using a temperature-sensitive DdAtg1, we
showed that DdAtg1 is required throughout development; development halts when
the cells are shifted to the restrictive temperature, but resumes when cells
are returned to the permissive temperature.
Submitted by: Richard Kessin [rhk2@columbia.edu]
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RacG regulates morphology, phagocytosis and chemotaxis
Baggavalli P. Somesh(1), Georgia Vlahou(1), Miho Iijima(3), Robert H.
Insall(2), Peter Devreotes(3) and Francisco Rivero(1)
1. Center for Biochemistry and Center for Molecular Medicine Cologne,
Medical Faculty, University of Cologne.
2. School of Biosciences, The University of Birmingham.
3. Department of Cell Biology, Johns Hopkins University School of Medicine.
Eukaryotic Cell, in press.
RacG is an unusual member of the complex family of Rho GTPses in
Dictyostelium. We have generated a knockout strain (KO) as well as strains
that overexpress the wild-type (WT), constitutively active (V12) or dominant
negative (N17) RacG. The protein is targeted to the plasma membrane,
apparently in a nucleotide-dependent manner, and induces the formation of
abundant actin-driven filopods. RacG is enriched at the rim of the
progressing phagocytic cup and overexpression of RacG-WT or RacG-V12 induced
an increased rate of particle uptake. The positive effect of RacG on
phagocytosis was abolished in the presence of 50 µM LY294002, a
phosphoinositide 3-kinase inhibitor, indicating that generation of
phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG.
RacG-KO cells showed a moderate chemotaxis defect that was stronger in
RacG-V12 and RacG-N17 mutants, in part due to interference with signaling
through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a
mutant lacking the Rho insert region, indicating that this region is
essential for interaction with downstream components. Processes like growth,
pinocytosis, exocytosis, cytokinesis and development were unaffected in
Rac-KO cells and in the overexpressor mutants. In a cell-free system RacG
induced actin polymerization upon GTPgammaS stimulation, and this response
could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO
cells indicates some overlap with one or more Dictyostelium Rho GTPases,
like Rac1 and RacB, the significant changes found in overexpressors show
RacG plays important roles. We hypothesize that RacG interacts with a subset
of effectors, in particular those concerned with shape, motility and
phagocytosis.
Submitted by: Francisco Rivero [francisco.rivero@uni-koeln.de]
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[End dictyNews, volume 27, number 5] 
