dictyNews
Electronic Edition
Volume 35, number 9
October 8, 2010
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Abstracts
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Coupling Mechanism of a GPCR and a Heterotrimeric G Protein During
Chemoattractant Gradient Sensing in Dictyostelium
Xuehua Xu,1*† Tobias Meckel,1*‡ Joseph A. Brzostowski,2 Jianshe Yan,
1 Martin Meier-Schellersheim,3 Tian Jin1†
1Chemotaxis Signal Section, National Institutes of Health, Rockville,
MD 20852, USA.
2Laboratory of Immunogenetics Imaging Facility, Laboratory of
Immunogenetics, National Institutes of Health, Rockville, MD 20852, USA.
3Program in Systems Immunology and Infectious Disease Modeling,
National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Rockville, MD 20852, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: [log in to unmask]
‡Present address: Department of Biology, Technische Universität Darmstadt,
Schnittspahnstrasse 3-5, D-64287 Darmstadt, Germany.
Science Signaling, Volume 3 Issue 141 ra71
The coupling of heterotrimeric guanine nucleotide–binding protein
(G protein)–coupled receptors (GPCRs) with G proteins is fundamental
for GPCR signaling; however, the mechanism of coupling is still debated.
Moreover, how the proposed mechanisms affect the dynamics of
downstream signaling remains unclear. Here, through experiments
involving fluorescence recovery after photobleaching and
single-molecule imaging, we directly measured the mobilities of cyclic
adenosine monophosphate (cAMP) receptor 1 (cAR1), a chemoattractant
receptor, and a G protein bg subunit in live cells. We found that cAR1
diffused more slowly in the plasma membrane than did Gbg. Upon
binding of ligand to the receptor, the mobility of cAR1 was unchanged,
whereas the speed of a fraction of the faster-moving Gbg subunits
decreased. Our measurements showed that cAR1 was relatively
immobile and Gbg diffused freely, suggesting that chemoattractant-bound
cAR1 transiently interacted with G proteins. Using models of possible
coupling mechanisms, we computed the temporal kinetics of G protein
activation. Our fluorescence resonance energy transfer imaging data
showed that fully activated cAR1 induced the sustained dissociation of
G protein a and bg subunits, which indicated that ligand-bound cAR1
activated G proteins continuously. Finally, simulations indicated that a
high-affinity coupling of ligand-bound receptors and G proteins was
essential for cAR1 to translate extracellular gradient signals into
directional cellular responses.We suggest that chemoattractant
receptors use a ligand-induced coupling rather than a precoupled
mechanism to control the activation of G proteins during chemotaxis.
Submitted by Xuehua Xu [[log in to unmask]]
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14-3-3 coordinates microtubules, Rac, and myosin II to control cell
mechanics and cytokinesis
Qiongqiong Zhou, Yee-Seir Kee, Christopher C. Poirier, Christine
Jelinek, Jonathan Osborne, Srikanth Divi, Alexandra Surcel,
Marie E. Will, Ulrike S. Eggert, Annette Müller-Taubenberger,
Pablo A. Iglesias, Robert J. Cotter, and Douglas N. Robinson
Curr. Biol., In press
Background: During cytokinesis, regulatory signals are presumed to
emanate from the mitotic spindle. However, what these signals are
and how they lead to the spatiotemporal changes in the cortex structure,
mechanics, and regional contractility are not well understood in any
system.
Results: To investigate pathways that link the microtubule network to
the cortical changes that promote cytokinesis, we used chemical
genetics in Dictyostelium to identify genetic suppressors of nocodazole,
a microtubule depolymerizer. We identified 14-3-3 and found that it
is enriched in the cortex, helps maintain steady state microtubule length,
contributes to normal cortical tension, modulates actin wave formation,
and controls the symmetry and kinetics of cleavage furrow contractility
during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac
small GTPase (RacE), associates with myosin II heavy chain and is
needed to promote myosin II bipolar thick filament remodeling.
Conclusion: 14-3-3 connects microtubules, Rac and myosin II to
control several aspects of cortical dynamics, mechanics, and
cytokinesis cell shape change. Further, 14-3-3 interacts directly
with myosin II heavy chain to promote bipolar thick filament r
emodeling and distribution. Overall, 14-3-3 appears to integrate
several critical cytoskeletal elements that drive two important
processes – cytokinesis shape change and cell mechanics.
Submitted by Doug Robinson [[log in to unmask]]
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The Dictyostelium discoideum acaA gene is transcribed from
alternative promoters during aggregation and multicellular
development.
Maria Galardi-Castilla, Ane Garciandía, Teresa Suarez and
Leandro Sastre
PloS one, in press
Background
Extracellular cAMP is a key extracellular signaling molecule that
regulates aggregation, cell differentiation and morphogenesis
during multi-cellular development of the social amoeba Dictyostelium
discoideum. This molecule is produced by three different adenylyl
cyclases, encoded by the genes acaA, acrA and acgA, expressed at
different stages of development and in different structures.
Methodology/Principal findings
This article describes the characterization of the promoter region
of the acaA gene, showing that it is transcribed from three different
alternative promoters. The distal promoter, promoter 1, is active during
the aggregation process while the more proximal promoters are active
in tip-organiser andposterior regions of the structures. A DNA fragment
containing the three promoters drove expression to these same regions
and similar results were obtained by in situ hybridization. Analyses of
mRNA expression by quantitative RT-PCR with specific primers for
each of the three transcripts also demonstrated their different
temporal patterns of expression.
Conclusions/Significance
The existence of an aggregation-specific promoter can be associated
with the use of cAMP as chemo-attractant molecule, which is specific
for some Dictyostelium species. Expression at late developmental
stages indicates that adenylyl cyclase A might play a more important
role in post-aggregative development than previously considered.
Submitted by Leandro Sastre [[log in to unmask]]
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Role of Magnesium and a Phagosomal P-type ATPase in
Intracellular Bacterial Killing
Emmanuelle Lelong, Anna Marchetti, Aurélie Guého,
Wanessa C. Lima, Natascha Sattler, Maëlle Molmeret,
Monica Hagedorn, Thierry Soldati and Pierre Cosson
Cellular Microbiology, in press
Bacterial ingestion and killing by phagocytic cells are essential
processes to protect the human body from infectious microorganisms.
However, only few proteins implicated in intracellular bacterial killing
have been identified to date. We used Dictyostelium discoideum,
a phagocytic bacterial predator, to study intracellular killing. In a
random genetic screen we identified Kil2, a type V P-ATPase as
an essential element for efficient intracellular killing of Klebsiella
pneumoniae bacteria. Interestingly, kil2 knockout cells still killed
efficiently several other species of bacteria, and did not show
enhanced susceptibility to Mycobacterium marinum intracellular
replication. Kil2 is present in the phagosomal membrane, and its
structure suggests that it pumps cations into the phagosomal lumen.
he killing defect of kil2 knockout cells was rescued by the addition
of magnesium ions, suggesting that Kil2 may function as a magnesium
pump. In agreement with this, kil2 mutant cells exhibited a specific
defect for growth at high concentrations of magnesium. Phagosomal
protease activity was lower in kil2 mutant cells than in wild-type cells,
a phenotype reversed by the addition of magnesium to the medium.
Kil2 may act as a magnesium pump maintaining magnesium
concentration in phagosomes, thus ensuring optimal activity of
phagosomal proteases and efficient killing of bacteria.
Submitted by Emmanuelle Lelong [[log in to unmask]]
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[End dictyNews, volume 35, number 9]
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