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dictyNews Volume 24 Number 06
Dicty News
Electronic Edition
Volume 24, number 6
March 11, 2005
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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Computer-Assisted Reconstruction and Analysis of Filopod Formation and the
Role of Myosin II Heavy Chain Phosphorylation in Dictyostelium
Paul J. Heid, Jeremy Geiger, Deborah Wessels, Edward Voss and David R. Soll
W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences,
The University of Iowa, Iowa City, IA 52242
J Cell Science, in press
To investigate the role played by filopodia in the motility and chemotaxis of
amoeboid cells, a computer-assisted 3D reconstruction and motion analysis
system, DIAS 4.0, has been developed. Reconstruction at short time intervals
of Dictyostelium amoebae migrating in buffer or in response to chemotactic
signals revealed 1) that the great majority of filopodia form on pseudopodia,
not on the cell body; 2) that filopodia on the cell body originate primarily
on pseudopodia and relocate; and 3) that filopodia on the uropod are longer
and more stable than those located on other portions of the cell. When
adjusting direction through lateral pseudopod formation in a spatial gradient
of chemoattractant, the temporal and spatial dynamics of lateral pseudopodia
suggest that filopodia may be involved in stabilizing pseudopodia on the
substratum while the decision is being made by a cell either to turn into a
pseudopodium formed in the correct direction (up the gradient) or to retract
a pseudopodium formed in the wrong direction (down the gradient). Experiments
in which amoebae were treated with high concentrations of chemoattractant
further revealed that receptor occupancy plays a role both in filopod
formation and retraction. Since phosphorylation-dephosphorylation of myosinII
heavy chain (MHC) plays a role in lateral pseudopod formation, turning and
chemotaxis, the temporal and spatial dynamics of filopod formation were
analyzed in MHC phosphorylation mutants. These latter studies revealed that
MHC phosphorylation-dephosphorylation plays a role in the regulation of
filopod formation during cell migration in buffer and during chemotaxis.
The computer-assisted technology described here for reconstructing filopodia
at short time intervals in living cells, therefore, provides a new tool for
investigating the role filopodia play in the motility and chemotaxis of
amoeboid cells.
Submitted by: Deborah Wessels [deborah-wessels@uiowa.edu]
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Dynamic properties of Legionella-containing phagosomes in Dictyostelium
amoebae
Hao Lu and Margaret Clarke
(Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical
Research Foundation, Oklahoma City, OK 73121)
Cellular Microbiology, in press
The natural hosts of the bacterial pathogen Legionella pneumophila are
amoebae and protozoa. In these hosts, as in human macrophages, the pathogen
enters the cell through phagocytosis, then rapidly modifies the phagosome to
create a compartment that supports its replication. We have examined L.
pneumophila entry and behavior during early stages of the infection of
Dictyostelium discoideum amoebae. Bacteria were labeled with a red
fluorescent marker, and selected proteins and organelles in the host were
labeled with GFP, allowing the dynamics and interactions of L. pneumophila-
containing phagosomes to be tracked in living cells. These studies
demonstrated that entry of L. pneumophila is an actin-mediated process, that
the actin-binding protein coronin surrounds the nascent phagosome but
dissociates immediately after internalization, that ER membrane is not
incorporated into a phagosome during uptake, that the newly internalized
phagosome is rapidly transported about the cell on microtubules, that
association of ER markers with the phagosome occurs in two steps that
correlate with distinct changes in phagosome movement, and that the
vacuolar H(+)-ATPase does not associate with mature replication vacuoles.
These studies have clarified certain aspects of the infection process
and provided new insights into the dynamic interactions between the
pathogen and its host.
Submitted by: Margaret Clarke [clarkem@omrf.ouhsc.edu]
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Manifestations of multicellularity - Dictyostelium reports
Jeffrey G. Williams*, Angelika A. Noegel+ and Ludwig Eichinger+
*School of Life Sciences
University of Dundee
MSI/WTB Complex
Dow Street
Dundee
DD1 5EH
U. K.
+ Centre for Biochemistry and Centre for Molecular Medicine Cologne
Medical Faculty, University of Cologne
Joseph-Stelzmann-Str. 52
50931 Cologne
Germany
TIGS Ms, in press
Foreword
The Dictyostelium genome sequence, the first complete amoebozoan sequence
to be released, has revealed many important evolutionary insights; notable
among these is the high degree of overlap between metazoan and Dictyostelium
proteomes.
Abstract
The recent release of the Dictyostelium genome sequence is important because
Dictyostelium has become a much-favoured model system for cell and
developmental biologists. The sequence has revealed a remarkably high total
number of app. 12,500 genes, only a thousand fewer than are encoded by
Drosophila. Previous protein sequence comparisons suggested that
Dictyostelium is evolutionary closer to animals and fungi than to plants
and the global protein sequence comparison, now made possible by the genome
sequence, confirms this. This review focuses on several classes of proteins
that are shared by Dictyostelium and animals: a highly sophisticated array
of microfilament components, a large family of G protein-coupled receptors
and a diverse set of SH2 domain-containing proteins. The presence of these
proteins strengthens the case for a relatively close relationship with
animals and extends the range of problems that can be addressed using
Dictyostelium as a model organism
Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk]
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cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum
Daniel F. Lusche, Karen Bezares-Roder, Kathrin Happle, Christina Schlatterer
Faculty for Biology, University of Konstanz, 78457 Konstanz, Germany
BMC Cell Biology, in press
Abstract
Background: Differentiating Dictyostelium discoideum amoebae respond upon
cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration
([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular
Ca2+-influx. In this study we investigated whether intracellular cAMP is
involved in the control of [Ca2+]i.
Results: We analyzed Ca2+-fluxes in a mutant that is devoid of the main
cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism.
In suspensions of developing cells cAMP-activated influx of extracellular
Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging
of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the
absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE
inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal
[Ca2+]i in both, mutant and wild type cells. Under this condition wild type
cells displayed cAMP-activated [Ca2+]i-transients also in nominally
Ca2+-free medium. In the mutant strain the amplitude of light scattering
oscillations and of accompanying cAMP oscillations were strongly reduced
to almost basal levels. In addition, chemotactic performance during
challenge with a cAMP-filled glass capillary was altered by EGTA-incubation.
Cells were more sensitive to EGTA treatment than wild type: already at
2 mM EGTA only small pseudopods were extended and chemotactic speed was
reduced.
Conclusions: We conclude that there is a link between the second messengers
cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this
link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of
regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating
Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering
oscillations and chemotactic performance.
Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de]
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Ca2+-regulation in the absaence of the iplA gene product in Dictyostelium
discoideum
Ralph H Schaloske 1, Daniel F Lusche 2, Karen Bezares-Roder 2,
Kathrin Happle 2, Dieter Malchow 2 and Christina Schlatterer 2
1 Department of Chemistry and Biochemistry, University of California at
San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601, USA
2 Faculty for Biology, University of Konstanz, 78457 Konstanz, Germany
BMC Cell Biology, in press
Abstract
Background
Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of
the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is
composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The
significance of the [Ca2+]i-transient for chemotaxis is under debate.
Abolition of chemotactic orientation and migration by Ca2+-buffers in the
cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet,
the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of
higher eukaryotes aggregates despite the absence of a cAMP-induced
[Ca2+]i-transient which favours the view that [Ca2+]i-changes are
insignificant for chemotaxis.
Results
We investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis
and development of iplA- cells. Differentiation was altered as compared to
wild type amoebae and sensitive towards manipulation of the level of stored
Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the
presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion
fluxes revealed that capacitative Ca2+-entry was fully operative in the
mutant. In suspensions of intact and permeabilized cells cAMP elicited
extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet
to a lesser extent than in wild type. In suspensions of partially purified
storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty
acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of
fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that
the responsiveness of mutant cells was shifted to higher concentrations:
roughly 100 times more Mn2+ was necessary to observe agonist-induced
Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly
loaded with Ca2+, again with a similar shift in sensitivity in the mutant.
In addition, basal [Ca2+]i was significantly lower in iplA- than in wild
type amoebae.
Conclusions
These results support the view that [Ca2+]i-transients are essential for
chemotaxis and differentiation. Moreover, capacitative and agonist-activated
ion fluxes are regulated by separate pathways that are mediated either by
two types of channels in the plasma membrane or by distinct mechanisms
coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The
iplA- strain retains the capacitative Ca2+-entry pathway and an impaired
agonist-activated pathway that operates with reduced efficiency or at
higher ionic pressure.
Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de]
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[End Dicty News, volume 24, number 6] 
