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dictyNews Volume 19 Number 14
Dicty News
Electronic Edition
Volume 19, number 14
December 13, 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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Calcium regulation of actin crosslinking is important for function of the
actin cytoskeleton in Dictyostelium
Ruth Furukawa, Andrew Maselli, Susanne A. M. Thomson, Rita W. L. Lim,
John V. Stokes and Marcus Fechheimer
Department of Cellular Biology, University of Georgia, Athens, Georgia
30602, USA
Journal of Cell Science In Press
The actin cytoskeleton is sensitive to changes in calcium,which affect
contractility, actin-severing proteins, actin-crosslinking proteins and
calmodulin-regulated enzymes. To dissect the role of calcium control on
the activity of individual proteins from effects of calcium on other
processes, calcium-insensitive forms of these proteins were prepared and
introduced into living cells to replace a calcium-sensitive form of the
same protein. Crosslinking and bundling of actin filaments by the
Dictyostelium 34 kDa protein is inhibited in the presence of micromolar
free calcium. A modified form of the 34 kDa protein with mutations in the
calcium binding EF hand (34 kDa DEF2) was prepared using site-directed
mutagenesis and expressed in E. coli. Equilibrium dialysis using
[ 45 Ca]CaCl2 revealed that the wild-type protein is able to bind one
calcium ion with a Kd of 2.4 mM. This calcium binding is absent in the 34
kDa DEF2 protein. The actin-binding activity of the 34 kDa DEF2 protein
was equivalent to wildtype but calcium insensitive in vitro. The wild-type
and 34 kDa DEF2 proteins were expressed in 34-kDa-null and 34 kDa/a-actinin
double null mutant Dictyostelium strains to test the hypothesis that calcium
regulation of actin crosslinking is important in vivo. The 34 kDa DEF2
failed to supply function of the 34 kDa protein important for control of
cell size and for normal growth to either of these 34-kDa-null strains.
Furthermore, the distribution of the 34 kDa protein and actin were abnormal
in cells expressing 34 kDa DEF2. Thus, calcium regulation of the formation
and/or dissolution of crosslinked actin structures is required for dynamic
behavior of the actin cytoskeleton important for cell structure and growth.
submitted by: amaselli@uconnvm.uconn.edu
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Constitutively Active Protein Kinase A Disrupts
Motility and Chemotaxis in Dictyostelium
Hui Zhang*, Paul J. Heid*, Deborah Wessels*, Karla J. Daniels*,
Tien Pham*, William F. Loomis?, and David R. Soll*?
*W.M. Keck Dynamic Image Analysis Facility
Department of Biological Sciences
University of Iowa
Iowa City, IA 52242
?Department of Biology
University of California, San Diego
La Jolla, CA 92039
Eukaryotic Cell, in press.
ABSTRACT
Deletion of the gene for the regulatory subunit of PKA results in
constitutively active PKA in the mutant pkaR-. To investigate the role
of
PKA in the basic motile behavior and chemotaxis of Dictyostelium, pkaR-
cells were subjected to computer-assisted 2D and 3D motion
analysis. pkaR- cells crawled at only half the speed of wild type cells
in buffer, chemotaxed in spatial gradients of cAMP, but with reduced
efficiency, were incapable of suppressing lateral pseudopods in the
front of temporal waves of cAMP, a requirement for natural chemotaxis,
did not exhibit the normal velocity surge in response to the front of a
wave, and were incapable of chemotaxing towards an aggregation
center in natural waves generated by a majority of wild type cells in
mixed cultures. Many of the behavioral defects appeared to be the
result of the constitutively ovoid shape of pkaR- cells, which forced
the dominant pseudopod off the substratum and to the top of the cell
body. The behavioral abnormalities that pkaR- cells shared with regA-
cells are discussed in considering the pathway ERK2 | RegA |
[cAMP] PKA, which emanates from the front of a wave. The results
demonstrate that cells must suppress PKA activity in order to
elongate along a substratum, suppress lateral pseudopod formation, and
crawl and chemotax efficiently. The results also implicate PKA
activation in dismantling cell polarity at the peak and in the back of a
natural cAMP wave.
submitted by: Hui Zhang [hui-zhang@uiowa.edu]
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Co-occurrence in nature of different clones of the social amoeba,
Dictyostelium discoideum
Angelo Fortunato, Joan E. Strassmann, Lorenzo Santorelli, and David C.
Queller
Department of Ecology and Evolutionary Biology MS170, Rice University, PO
Box 1892, Houston, Texas, 77251-1892, USA
Molecular Ecology, in press
Abstract.
The social amoeba, Dictyostelium discoideum, produces a multicellular
fruiting body and has become a model system for cell-cell interactions such
as signaling, adhesion, and development. However, unlike most multicellular
organisms, it forms by aggregation of cells and, in the lab, it forms
genetic chimeras where there may be competition among clones. Here we
show that chimera formation is also likely in nature, because different
clones commonly co-occur on a very small scale. This suggests that D.
discoideum will likely have evolved strategies for competing in chimeras,
and that the function of some developmental genes will be competitive.
Natural chimerism also makes D. discoideum a good model organism for the
investigation of issues relating to coexistence and conflict between cells.
submitted by: David Queller [queller@pop.ruf.rice.edu]
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A diverse family of inositol 5-phosphatases playing a role in growth and
development in Dictyostelium discoideum
Harrit M. Loovers, Kees Veenstra, Helena Snippe, Xavier Pesesse,
Christophe Erneux, and Peter J.M. van Haastert
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747
AG Groningen, the Netherlands and
Institute of Interdisciplinary Research, Free University of Brussels,
Campus Erasme, Bldg. C, 808 Route de Lennik, B-1070 Brussels, Belgium.
J. Biol. Chem, in press
Inositol phosphate containing molecules play an important role in a broad
range of cellular processes. Inositol 5-phosphatases participate in the
regulation of these signaling molecules. We have identified four inositol
5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity
in domain composition. Dd5P1 possesses only a inositol 5-phosphatase
catalytic domain. An unique domain composition is present in Dd5P2,
containing a RCC1-like domain; RCC1 has a seven-bladed propeller structure
and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition
similar to human Synaptojanin, with a Sac1 domain, and OCRL, with a RhoGAP
domain, respectively. We have expressed the catalytic domains and show that
these inositol 5-phosphatases have different substrate preferences. Single
and double gene inactivation suggest a functional redundancy for Dd5P1,
Dd5P2 and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to
defects in growth and development. These defects are restored by expression
of the complete protein, but not by the 5-phosphatase catalytic domain.
submitted by: P.J.M.van.Haastert [P.J.M.van.Haastert@chem.rug.nl]
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[End Dicty News, volume 19, number 14]
