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dictyNews Volume 17 Number 02
Dicty News
Electronic Edition
Volume 17, number 2
July 14, 2001
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.
===================
Postoc Position
===================
Postdoctoral position (BATIIa) in Molecular Genetics at Kassel University
(3 years + 2 years).
A postdoctoral position is available to work on RNA mediated gene silencing
(antisense RNA, RNAi) in Dictyostelium or on signal transduction in early
development (see http://www.uni-kassel.de/fb19/genetics/ for detailed
information). The laboratory is well equipped for molecular biology. In
addition, scanning force microscopy on RNA-protein interactions is
established. Cooperations with others groups in the Biology Department are
encouraged.
The successful applicant should have solid experience in molecular biology
and/or protein biochemistry. The development of an independent reseach
program along the lines of the general laboratory interests and the
establishment of a small research group are encouraged. Modest
participation in teaching biology students is required. Applicants should
attempt the habilitation or an equivalent qualification. Interested
individuals may first contact W. Nellen (nellen@hrz.uni-kassel.de) by
e-mail or dirctly send applications to
Prsident
Universitt Kassel
D-34109 Kassel
Germany
Kennziffer 1114
==============
Abstracts
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Under-Agarose Folate Chemotaxis of Dictyostelium Amoebae in Permissive and
Mechanically Inhibited Conditions
Gary Laevsky and David A. Knecht
Department of Molecular and Cell Biology, University of Connecticut, Storrs,
CT 06269
in Press: Biotechniques
ABSTRACT
Under agarose chemotaxis has been used previously to assess the
ability of neutrophils to respond to gradients of chemoattractant. We have
adapted this assay to the chemotactic movement of Dictyostelium amoebae in
response to folic acid. Troughs are used instead of wells in order to increase
the area along which the cells can be visualized and to create a uniform
front of moving cells. Imaging the transition zone where the cells first
encounter the agarose, we find that the cells move perpendicular to the
gradient and periodically manage to squeeze under the agarose and move up
the gradient. As cells exit the troughs, their cross sectional area
increases as the cells become flattened. Three-dimensional reconstruction
of confocal optical sections through GFP labeled-cells demonstrates that
the increase in cross sectional area is due to the flattening of the cells.
Since the cells locally deform the agarose and become deformed by it, the
concentration of the agarose and therefore its stiffness should affect the
ability of the cells to migrate. Consistent with this hypothesis, cells
in 0.5% agarose move faster and are less flat than cells under 2% agarose.
Cells do not exit the troughs and move under 3% agarose at all. Therefore
this assay can be used to compare and quantify the ability of different
cells types or mutant cell lines to move in a restrictive environment.
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Automated Real-time Measurement of Chemotactic Cell Motility.
Nacima Hadjout, Gary Laevsky, David A. Knecht, and Michael A. Lynes
Department of Molecular and Cell Biology, University of Connecticut
Storrs, CT 06269-3125
in Press: Biotechniques
ABSTRACT
We have developed a novel method (ECIS/taxis) for monitoring cell
movement in response to chemotactic and chemokinetic factors. In this system,
cells migrate in an under-agarose environment and their position is monitored
using the Electric Cell Impedance Sensor (ECIS) technology to measure the
impedance change at a target electrode lithographed onto the substrate as the
cells arrive at that target. In the studies reported here, Dictyostelium
discoideum was used as a prototypical motile eukaryotic cell. Using the
ECIS/taxis system, the arrival of cells at the target electrode was proportional
to the dose of folate used to stimulate the cells and could be assessed by
changes in resistance at the electrode. ECIS/taxis was readily able to
distinguish between wild-type cells and a mutant that is deficient in its
chemotactic response. Finally, we have shown that an agent that interferes
with chemotactic motility leads to delayed arrival of cells at the target
electrode. The multiwell configuration of the assay allows for
simultaneous automated screening of many samples for chemotactic or
anti-chemotactic activity. This assay system is compatible with measurements
of mammalian cell movement and should be valuable in the assessment of both
agonists and antagonists of cell movement.
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Reduced protein diffusion rate by cytoskeleton in vegetative and polarized
Dictyostelium cells
Eric O. Potma, Wim P. de Boeij, Leonard Bosgraaf, Jeroen Roelofs, Peter
J. M. Van Haastert and Douwe A Wiersma
Ultrafast Laser and Spectroscopy Laboratory and Department
of Biochemistry, University of Groningen, Nijenborgh 4, 9747
AG Groningen, the Netherlands
Biophysical Journal, in press
Fluorescence recovery after photobleaching measurements with
high spatial resolution are performed to elucidate the impact of
the actin cytoskeleton on translational mobility of green
fluorescent protein (GFP) in aqueous domains of Dictyostelium
discoideum amoebae. In vegetative Dictyostelium cells, GFP
molecules experience a 3.6 fold reduction of their translational
mobility relative to dilute aqueous solutions. In disrupting the
actin filamentous network using latrunculin-A, the intact actin
cytoskeletal network is shown to contribute an effective viscosity
of 1.36 cP, which accounts for 53% of the restrained molecular
diffusion of GFP. The remaining 47% of hindered protein
motions is ascribed to other mechanical barriers and the
viscosity of the cell liquid. A direct correlation between the
density of the actin network and its limiting action on protein
diffusion is furthermore established from measurements under
different osmotic conditions. In highly locomotive polarized cells
the obstructing effect of the actin filamentous network is seen to
decline to 0.46 cP in the non-cortical regions of the cell. Our
results indicate that the meshwork of actin filaments constitutes
the primary mechanical barrier for protein diffusion and that any
noticeable reorganization of the network is accompanied with an
altered intracellular protein mobility.
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Recruitment of Cortexillin into the Cleavage Furrow is Controlled by Rac1
and IQGAP-related Proteins
Jan Faix 1, Igor Weber 2, Ursula Mintert 2, Jana Khler 2, Friedrich
Lottspeich 2, and Gerard Marriott 1
1) Department of Physiology, University of Wisconsin-Madison, 1300
University Ave, WI, 53706, USA.
2) Max-Planck-Institut fr Biochemie, D-82152 Martinsried, Germany.
EMBO Journal, Vol.20, pp3705-3715, 2001
Abstract
Cytokinesis in eukaryotic organisms is under the control of small
GTP-binding proteins, although the underlying molecular mechanisms are not
fully understood. Cortexillins are actin-binding proteins whose activity is
crucial for cytokinesis in Dictyostelium. Here we show that the
IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with
the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I,
DGAP1 is enriched in the cortex of interphase cells and translocates to the
cleavage furrow during cytokinesis. The activated form of the small GTPase
Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II.
In DGAP1- mutants, a complex can still be formed with a second
IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and
GAPA, however, prevents complex formation and localization of the
cortexillins to the cleavage furrow. This leads to a severe defect in
cytokinesis, which is similar to that found in cortexillin I/II double-null
mutants. Our observations define a novel and functionally significant
signaling pathway that is
required for cytokinesis.
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Sensing and responding to chemoattractants: Signaling pathways controlling
cell polarity and directional cell movement
Chang Y. Chung, Satoru Funamoto, and Richard A. Firtel
Trends in Biochemical Sciences (TiBS)
Summary
Many important biological processes, including chemotaxis, or directional
cell movement up a gradient of a chemoattractant, require the ability to
respond to a directional signal and a clearly established cell polarity.
Recent advances using Dictyostelium cells and mammalian leukocytes have
provided insights into the biochemical and molecular pathways that control
chemotaxis. Phosphoinositide 3-kinase (PI3K) plays a central and possibly
pivotal role in establishing and maintaining cell polarity by regulating
the subcellular localization and activation of downstream effectors that
are essential for regulating cell polarity and proper chemotaxis. This
review outlines our present understanding of these pathways.
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Dictyostelium centrin-related protein (DdCrp), the most divergent member of
the centrin family, possesses only two EF-hands and dissociates from the
centrosome during mitosis.
Christine Daunderer, Manfred Schliwa and Ralph Grf1)
Adolf-Butenandt-Insitut/Zellbiologie, Universitt Mnchen, Germany
Eur. J. Cell Biol, in press
We have identified a Dictyostelium discoideum cDNA sequence with homology
to centrins. The derived protein, Dictyostelium discoideum centrin-related
protein (DdCrp), is the most divergent member of the centrin family. Most
strikingly it lacks the first two EF-hand consensus motifs, whereas a
number of other centrin-specific sequence features are conserved. Southern
and Northern blot analysis and the data presently available from the
Dictyostelium genome and cDNA projects suggest that DdCrp is the only
centrin isoform present in Dictyostelium. Immunofluorescence with
anti-DdCrp antibodies revealed that the protein is localized to the
centrosome, to a second, centrosome-associated structure close to the
nucleus and to the nucleus itself. Confocal microscopy resolved that the
centrosomal label is confined to the corona surrounding the centrosome
core. Unlike for other centrins the localization of DdCrp is cell-cycle
dependent. Both the centrosomal and the centrosome associated label
disappear during prometaphase, most likely in concert with the dissociation
of the corona at this stage. The striking differences of DdCrp to all other
centrins may be related to the distinct structure and duplication mode of
the Dictyostelium centrosome.
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[End Dicty News, volume 17, number 2]
