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dictyNews Volume 23 Number 13
Dicty News
Electronic Edition
Volume 23, number 13
October 15, 2004
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 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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In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium
phagocytosis and chemotaxis
Dirk Dormann (1), Gerti Weijer (1), Simon Dowler (2) and Cornelis
J. Weijer (1)
1. Division of Cell and Developmental Biology, MSI/WTB Complex,
University of Dundee, Dow Street, Dundee DD1 5EH, UK; 2. BioFocus Ltd,
Cambridge, UK
Journal of Cell Science, in press
Phagocytosis and chemotaxis are receptor-mediated processes that require
extensive rearrangements of the actin cytoskeleton, and are controlled by
lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate
[PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]. We
used a panel of Pleckstrin Homology (PH) domains with distinct binding
specificities for PI(3,4,5)P3 and PI(3,4)P2 to study the spatiotemporal
dynamics of these phosphoinositides in vivo. During phagocytosis and
macropinocytosis PI(3,4,5)P3 levels transiently increase at sites of
engulfment, followed by a rapid PI(3,4)P2 production round the
phagosome/macropinosome upon its internalisation, suggesting that
PI(3,4,5)P3 is degraded to PI(3,4)P2. PTEN null mutants, which are
defective in phagocytosis, show normal rates of PI(3,4,5)P3 degradation,
but unexpectedly an accelerated PI(3,4)P2 degradation. During chemotaxis
to cAMP only PI(3,4,5)P3 is formed in the plasma membrane, no PI(3,4)P2
is detectable showing, that all PI(3,4,5)P3 is degraded by PTEN to
PI(4,5)P2. We furthermore show that different PI(3,4,5)P3 binding PHdomains
give distinct spatial and temporal readouts of the same underlying
PI(3,4,5)P3 signal, enabling distinct biological responses to one signal.
Submitted by: Dirk Dormann [d.dormann@dundee.ac.uk]
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Exposure of cells to a cell-number counting factor decreases the activity of
glucose-6-phosphatase to decrease intracellular glucose levels in
Dictyostelium
Wonhee Jang and Richard H. Gomer
Howard Hughes Medical Institute and Department of Biochemistry and Cell
Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892
Eukaryotic Cell, in press
The development of Dictyostelium discoideum is a model for tissue size
regulation as these cells form groups of ~2 x 104 cells. The group size
is regulated in part by a negative feedback pathway mediated by a secreted
multi-polypeptide complex called counting factor (CF). CF signal
transduction involves CF decreasing intracellular glucose levels. A
component of CF, countin, has the bioactivity of the entire CF complex, and
an 8-minute exposure of cells to recombinant countin decreases intracellular
glucose levels. To understand how CF regulates intracellular glucose, we
examined the effect of CF on enzymes involved in glucose metabolism.
Exposure of cells to CF has little effect on amylase or glycogen
phosphorylase, enzymes involved in glucose production from glycogen.
Glucokinase activity (the first specific step of glycolysis) is inhibited
by high levels of CF but is not affected by an 8-minute exposure to countin.
The second enzyme specific for glycolysis, phosphofructokinase, is not
regulated by CF. There are two corresponding enzymes in the gluconeogenesis
pathway, fructose-1,6-bisphosphatase and glucose-6-phosphatase. The first
is not regulated by CF or countin, whereas glucose-6-phosphatase is
regulated by both CF and an 8-minute exposure to countin. The
countin-induced changes in the Km and Vmax of glucose-6-phosphatase cause a
decrease in glucose production that can account for the countin-induced
decrease in intracellular glucose levels. It thus appears that part of the
CF signal transduction pathway involves inhibiting the activity of
glucose-6-phosphatase, decreasing intracellular glucose levels and affecting
the levels of other metabolites, to regulate group size.
Submitted by: Richard Gomer [richard@rice.edu]
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Cellular distribution and functions of wild-type and constitutively activated
Dictyostelium PakB
Marc de la Roche, Amjad Mahasneh, Sheu-Fen Lee, Francisco Rivero* and
Graham P. Ct¤
Department of Biochemistry, Queen's University, Kingston, Ontario,
Canada K7L 3N6; *Zentrum fr Biochemie, Medizinische Fakultt, Universitt
zu Kln, Joseph-Stelzmann-Strasse 52, 50931 Kln, Germany
Molecular Biology of the Cell, In Press
Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a
member of the p21-activated kinase (PAK) family. Two-hybrid assays showed
that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein),
RacB, RacC and RacF1. Wild-type PakB displayed a cytosolic distribution
with a modest enrichment at the leading edge of migrating cells and at
macropinocytic and phagocytic cups, sites consistent with a role in
activating myosin I. PakB fused at the N-terminus to GFP was proteolyzed
in cells, resulting in removal of the catalytic domain. C-terminal
truncated PakB and activated PakB lacking the p21-binding domain strongly
localized to the cell cortex, to macropinocytic cups, to the posterior of
migrating cells and to the cleavage furrow of dividing cells. These data
indicate that in its open, active state, the N-terminus of PakB forms a
tight association with cortical actin filaments. PakB-null cells displayed
no significant behavioral defects, but cells expressing activated PakB were
unable to complete cytokinesis when grown in suspension and exhibited
increased rates of phagocytosis and pinocytosis.
Submitted by: Graham Cote [coteg@post.queensu.ca]
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[End Dicty News, volume 23, number 13] 
