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dictyNews Volume 31 Number 02
dictyNews
Electronic Edition
Volume 31, number 2
July 11, 2008
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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The GPI-anchored Superoxide Dismutase, SodC, is essential for regulating
basal Ras activity and chemotaxis of Dictyostelium discoideum
Sudhakar Veeranki, Bohye Kim, and Leung Kim*
Dept of Biological Sciences, Florida International University,
Miami, Fl 33199 USA
Journal of Cell Sciences, in press
A genetic screen for Dictyostelium mutant displaying high level of constitutive
Phosphatidylinositol-3,4,5-triphosphate (PIP3) led to the finding that the
glycosylphosphatidylinositol (GPI) anchored superoxide dismutase, SodC,
regulates small GTPase Ras. Cells lacking SodC exhibited constitutively high
levels of active Ras, more membrane localization of GFP-PHcrac, and defects in
chemoattractant sensing, cell polarization, and motility. These defects of sodC-
cells were partially restored by expression of wild type SodC but not with the
catalytically inactive mutant SodC (H245R, H247Q). Furthermore, an inhibition of
PI3K activity in sodC- cells by LY294002 only partially restored chemoattractant
sensing and cell polarization, consistent with the fact that sodC- cells have
aberrantly high level of active Ras, which functions upstream of PI3K. Higher
level of active GFP-RasG was observed in sodC- cells, which significantly
decreased upon incubation of sodC- cells with the superoxide scavenger XTT.
Having constitutively high levels of active Ras proteins and more membrane
localization of GFP-PHcrac, sodC- cells exhibited severe defects in
chemoattractant sensing, cell polarization, and motility.
Submitted by: Kim Leung [kiml@fiu.edu]
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Dictyostelium kinase DPYK3 negatively regulates STATc signaling in cell fate
decision.
Nam-Sihk Lee, Marbelys Rodriguez, Bohye Kim, and Leung Kim*
Department of Biological Sciences
Florida International University
Miami, FL 33199 USA
Development, Growth and Differentiation, in press
DPYK3, a member of the Dictyostelium TKL (Tyrosine Kinase Like) kinase family,
was ablated by homologous recombination. dpyk3- cells displayed aberrant
pattern formation during development. The prestalk O zone was not properly
formed and, instead, the prespore zone was expanded in dpyk3- slugs. During
development, the transcription factor STATc was persistently phosphorylated
and ecmAO expression level was kept low in dpyk3- cells. Furthermore, in
response to DIF-1 in suspension culture, dypk3- cells displayed persistent
STATc phosphorylation and re-introduction of DPYK3 in dypk3- cells restored
transient STATc phosphorylation similarly to wild type cells. In contrast to the
positive STAT regulation by Janus Kinase in metazoans, Dictyostelium DPYK3
negatively regulates STATc during development in response to DIF-1 signaling.
Submitted by: Kim Leung [kiml@fiu.edu]
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Modulation of actin structure and function by phosphorylation of Tyr-53
and profilin binding
Kyuwon Baek*†, Xiong Liu*‡, François Ferron†, Shi Shu‡, Edward D. Korn‡§,
Roberto Dominguez†§
†University of Pennsylvania School of Medicine, Department of Physiology,
3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
‡Laboratory of Cell Biology, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, MD 20892, USA.
*These authors contributed equally to this work
Proc. Natl. Acad. Sci. USA, in press
On starvation, Dictyostelium cells aggregate to form multicellular fruiting
bodies containing spores that germinate when transferred to nutrient-rich
medium. This developmental cycle correlates with the extent of actin
phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells
but high in viable mature spores. Here we describe high-resolution crystal
structures of pY53-actin and unphosphorylated actin in complexes with
gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate
group on Tyr-53 makes hydrogen-bonding interactions with residues
of the DNase I-binding loop (D-loop) of actin, resulting in a more stable
conformation of the D-loop than in the unphosphorylated structures. A more
rigidly folded D-loop may explain some of the previously described
properties of pY53-actin, including its increased critical concentration for
polymerization, reduced rates of nucleation and pointed end elongation, and
weak affinity for DNase I. We show here that phosphorylation of Tyr-53
inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide
exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly
similar to previously determined structures of profilin-beta-actin and
profilin-alpha-actin. By comparing this representative set of profilin-actin
structures with other structures of actin we highlight the effects of profilin
on the actin conformation. In the profilin-actin complexes, subdomains
1 and 3 of actin close around profilin, producing a 4.7º rotation of the two
major domains of actin relative to each other. As a result, the nucleotide cleft
becomes moderately more open in the profilin-actin complex, probably
explaining the stimulation of nucleotide exchange on actin by profilin.
Submitted by: Edward Korn [edk@nih.gov]
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Actin-Cytoskeleton Dynamics in Non-monotonic Cell Spreading
Doris Heinrich1, Simon Youssef1, Britta Schroth-Diez2, Ulrike Engel3,
Daniel Aydin4, Jacques Bluemmel4, Joachim Spatz4, and Guenther Gerisch5*
1 Department fuer Physik, Ludwig-Maximilians-University,
Geschwister-Scholl-Platz 1, D-80539 Muenchen, Germany.
2 Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstr. 108, D-01307 Dresden, Germany.
3 Nikon Imaging Center at the University of Heidelberg, Bioquant BQ 0004,
Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany.
4 Max Planck Institute for Metals Research, Dept. of New Materials and
Biosystems, Heisenbergstr. 3, D-70569 Stuttgart, Germany, and
University of Heidelberg, Dept. of Biophysical Chemistry.
5 Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18, D-82152
Martinsried, Germany.
Cell Adhesion & Migration, in press
The spreading of motile cells on a substrate surface is accompanied by
reorganization of their actin network. We show that spreading in the highly
motile cells of Dictyostelium is non-monotonic, and thus differs from the
passage of spreading cells through a regular series of stages. Quantification
of the gain and loss of contact area revealed fluctuating forces of protrusion
and retraction that dominate the interaction of Dictyostelium cells with a
substrate. The molecular basis of these fluctuations is elucidated by
dual-fluorescence labeling of filamentous actin together with proteins that
highlight specific activities in the actin system. Front-to-tail polarity is
established by the sorting out of myosin-II from regions where dense actin
assemblies are accumulating. Myosin-IB identifies protruding front regions,
and the Arp2/3 complex localizes to lamellipodia protruded from these regions.
Coronin is used as a sensitive indicator of actin disassembly to visualize
the delicate balance of polymerization and depolymerization in spreading
cells. Short-lived actin patches that co-localize with clathrin suggest that
membrane internalization occurs even when the substrate-attached cell
surface expands. We conclude that non-monotonic cell spreading is
characterized by spatiotemporal patterns formed by motor proteins together
with regulatory proteins that either promote or terminate actin polymerization
on the scale of seconds.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
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Regulation of Contractile Vacuole Formation and Activity in Dictyostelium
Fei Du, Kimberly Edwards, Zhouxin Shen, Binggang Sun, Arturo De Lozanne,
Steven Briggs, and Richard A. Firtel
Section of Cell and Developmental Biology, Division of Biological Sciences,
University of California San Diego, La Jolla, CA 92093-0380, USA
and Section of Molecular Cell & Developmental Biology and Institute for
Cellular and Molecular Biology, University of Texas at Austin,
Austin, TX 78712, USA
EMBO J., in press
The contractile vacuole (CV) system is the osmoregulatory organelle required
for survival for many free-living cells under hypotonic conditions. We
identified a new CV regulator, Disgorgin, a TBC domain-containing protein,
which translocates to the CV membrane at the late stage of CV charging and
regulates CV/plasma membrane fusion and discharging. disgorgin- cells produce
large CVs due to impaired CV/plasma membrane fusion. Disgorgin is a specific
GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is
required for discharging. We demonstrate that Drainin, a previously identified
TBC-domain-containing protein, lies upstream from Disgorgin in this pathway.
Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A
effector. The BEACH family proteins LvsA and LvsD were identified in a
suppressor/enhancer screen of the disgorgin- large CV phenotype and
demonstrated to have distinct functions in regulating CV formation. Our
studies help define the pathways controlling CV function.
Submitted by: Rick Firtel [rafirtel@ucsd.edu]
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Article Addendum
Vacuole membrane protein 1, autophagy and much more
Javier Calvo-Garrido, Sergio Carilla-Latorre and Ricardo Escalante
Instituto de Investigaciones Biomedicas Alberto Sols, Consejo Superior de
Investigaciones Científicas-Universidad Autonoma de Madrid,
Calle Arturo Duperier 4, 28029 Madrid, Spain
Autophagy, In press
Vacuole membrane protein 1 (Vmp1) is a putative transmembrane protein that
has been associated with different functions including autophagy, cell
adhesion and membrane traffic. Highly similar proteins are present in lower
eukaryotes and plants although a homologue is absent in the fungi lineage.
We have recently described the first loss-of-function mutation for a Vmp1
homologue in a model system, Dictyostelium discoideum. Our results give a
more comprehensive view of the intricate roles played by this new gene.
Dictyostelium Vmp1 is an endoplasmic reticulum-resident protein. Cells
deficient in Vmp1 display pleiotropic defects in the context of the
secretory pathway such as organelle biogenesis, the endocytic pathway and
protein secretion. The biogenesis of the contractile vacuole, an organelle
necessary to survive under hypoosmotic conditions, is compromised as well
as the structure of the endoplasmic reticulum and the Golgi apparatus.
Transmission electron microscopy also shows abnormal accumulation of
aberrant double-membrane vesicles, suggesting a defect in autophagosome
biogenesis or maturation. The expression of a mammalian Vmp1 in the
Dictyostelium mutant complements the phenotype suggesting a functional
conservation during evolution. We are taking the first steps in
understanding the function of this fascinating protein and recent studies
have brought us more questions than answers about its basic function and
its role in human pathology.
Submitted by: Ricardo Escalante [rescalante@iib.uam.es]
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Kinesin-5 is Not Essential for Mitotic Spindle Elongation in Dictyostelium
Irina Tikhonenko, Dilip K. Nag, Nora Martin, and Michael P. Koonce
Wadsworth Center, Albany, NY
Cell Motil. Cytoskel, in press
The proper assembly and operation of the mitotic spindle is essential to
ensure the accurate segregation of chromosomes and to position the cytokinetic
furrow during cell division in eukaryotes. Not only are dynamic microtubules
required, but also the concerted actions of multiple motor proteins are
necessary to effect spindle pole separation, chromosome alignment, chromatid
segregation, and spindle elongation. Although a number of motor proteins are
known to play a role in mitosis, there remains a limited understanding of
their full range of functions and the details by which they interact with
other spindle components. The kinesin-5 (BimC/Eg5) family of motors is
largely considered essential to drive spindle pole separation during the
initial and latter stages of mitosis. We have deleted the gene encoding the
kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast
with results found in vertebrate, fly, and yeast organisms, kif13- cells
continue to grow at rates indistinguishable from wild type. Phenotype analysis
reveals a slight increase in spindle elongation rates in the absence of Kif13.
More importantly, there is a dramatic, premature separation of spindle halves i
n kif13- cells, suggesting a novel role of this motor in maintaining spindle
integrity at the terminal stages of division.
Submitted by: Michael Koonce [koonce@wadsworth.org]
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[End dictyNews, volume 31, number 2]
