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dictyNews Volume 28 Number 10
dictyNews
Electronic Edition
Volume 28, number 10
April 20, 2007
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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Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced
actin polymerization
Paul D. Langridge and Robert R. Kay
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK
Experimental Cell Research, in press
We have investigated the role of the Arp2/3 complex in Dictyostelium cell
chemotaxistoward cyclic-AMP and in the actin polymerization that is triggered
by thischemoattractant. We confirm that the Arp2/3 complex is recruited to
the cell perimeter, orinto a pseudopod, after cyclic-AMP stimulation and that
this is coincident with actinpolymerization. This recruitment is inhibited
when actin polymerization is blocked usinglatrunculin suggesting that the
complex binds to pre-existing actin filaments, rather thanto a membrane
associated signalling complex. We show genetically that an intact
Arp2/3complex is essential in Dictyostelium and have produced partially
active mutants in twoof its subunits. In these mutants both phases of actin
polymerization in response to cyclic-AMP are greatly reduced. One mutant
projects pseudopodia more slowly than wild typeand has impaired chemotaxis,
together with slower movement. The second mutantchemotaxes poorly due to an
adhesion defect, suggesting that the Arp2/3 complex plays acrucial part in
adhering cells to the substratum as they move. We conclude that theArp2/3
complex largely mediates the actin polymerization response to
chemotacticstimulation and contributes to cell motility, pseudopod extension
and adhesion inDictyostelium chemotaxis.
Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk]
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An intracellular P2X receptor required for osmoregulation in Dictyostelium
discoideum
Samuel J. Fountain, Katie Parkinson, Mark T. Young, Lishuang Cao, Christopher
R.L. Thompson* and R. Alan North*
*Correseponding authors
Faculty of Life Sciences, University of Manchester, Michael Smith Building,
Oxford Road, Manchester M13 9PT, England, UK
Nature, in press
P2X receptors are membrane ion channels gated by extracellular ATP found
widely in vertebrates, but not previously in microbes. We identified a
weakly related gene in the genome of the social amoeba Dictyostelium
discoideum, and heterologous expression in HEK cells showed that it encoded
a membrane ion channel activated by ATP (30 Ð 100 mM). Site-directed
mutagenesis revealed essential conservation of structure-function relations
with P2X receptors of higher organisms. The receptor was insensitive to
usual P2X antagonists3 but blocked by nanomolar concentrations of copper.
In D. discoideum, the receptor was found on intracellular membranes, with
prominent localization to an osmoregulatory organelle, the contractile
vacuole. Targeted disruption of the gene in D. discoideum resulted in cells
unable to regulate cell volume in hypotonic conditions. Cell swelling in
these mutant cells was accompanied by a marked inhibition of contractile
vacuole emptying. These findings demonstrate a new functional role for P2X
receptors on intracellular organelles, in this case in osmoregulation.
Submitted by: Chris Thompson [christopher.thompson@man.ac.uk]
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Essential role of PI 3-kinase and phospholipase A2 in Dictyostelium chemotaxis
Peter J.M. van Haastert, Ineke Keizer-Gunnink and Arjan Kortholt
Department of Molecular Cell Biology, University of Groningen, Kerklaan 30,
9751NN Haren, the Netherlands
J. Cell Biology, in press
Chemotaxis towards different cAMP concentrations was tested in Dicyostelium
cell lines with deletion of specific genes together with drugs to inhibit one
or all combinations of the second messenger systems PI3-kinase, PLC, PLA2,
and cytosolic Ca2+. The results show that inhibition of either PI3-kinase or
PLA2 inhibits chemotaxis in shallow cAMP gradients, while both enzymes must
be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that
PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells
lacking PLC activity have normal chemotaxis; however, additional inhibition
of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has
no effect, suggesting that all chemotaxis in plc-null cells is mediated by
PLA2. Cells with deletion of the IP3-receptor have the opposite phenotype:
chemotaxis is completely dependent on PI3-kinase, and insensitive to
PLA2-inhibitors. This suggest that PI3-kinase-mediated chemotaxis is
regulated by PLC, probably through controlling PIP2 levels and PTEN
activity, whereas chemotaxis mediated by PLA2 appears to be controlled by
intracellular Ca2+.
Submitted by: Peter van Haastert [p.j.m.van.haastert@rug.nl]
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Biased random walk by stochastic fluctuations of chemoattractant-receptor
interactions at the lower limit of detection
Peter J.M. van Haastert and Marten Postma
Department of Molecular Cell Biology, University of Groningen, Kerklaan 30,
9751NN Haren, the Netherlands
Department of Physiology, Development and Neuroscience, Cambridge University,
Downing St, Cambridge CB2 3DY UK
Biophysical Journal, in press
Binding of ligand to its receptor is a stochastic process that exhibits
fluctuations in time and space. In chemotaxis this leads to a noisy input
signal. Therefore, in a gradient of chemoattractant the cell may occasionally
experience a ÒwrongÓ gradient of occupied receptors. We obtained a simple
equation for Ppos, the probability that half of the cell closest to the
source of chemoattractant has higher receptor occupancy than the opposite
half of the cell. Ppos depends on four factors, the gradient property
deltaC/sqrtC, the receptor characteristic Rt/Kd , a time-averaging constant I,
and non-receptor noise. We measured chemotaxis of Dictyostelium cells to known
shallow gradients of cAMP and obtained direct estimates for these constants.
Furthermore, we observed that in shallow gradients the measured chemotaxis
index is correlated with Ppos, which suggest that chemotaxis in shallow
gradients is a pure biased random walk. From the observed chemotaxis and
derived time-averaging constant we deduce that the gradient transducing second
messenger has a life time of 2 to 8 seconds and a diffusion rate constant of
~1 microm^2/s. Potential candidates for such second messengers are discussed.
Submitted by: Peter van Haastert [p.j.m.van.haastert@rug.nl]
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[End dictyNews, volume 28, number 10]
