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dictyNews Volume 21 Number 17
Dicty News
Electronic Edition
Volume 21, number 17
November 28, 2003
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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Generation of double gene disruptions in Dictyostelium discoideum using a
single antibiotic marker selection
Venkaiah Betapudi, Karen Shoebotham, and Thomas T. Egelhoff
Department of Physiology and Biophysics, Case Western Reserve University,
10900 Euclid Avenue, Cleveland, OH 44106, USA
Biotechniques, in press
Gene targeting is a powerful molecular genetic technique that has been widely
used to understand specific gene function in vivo. This technique allows the
ablation of an endogenous gene by recombination between an introduced DNA
fragment and the homologous target gene. However, when multiple gene
disruptions are needed, the availability of only a limited number of marker
genes becomes a complication. Here we describe a new approach to perform
double gene disruptions in Dictyostelium discoideum by simultaneous
transfection of two gene targeting cassettes followed by performing clonal
selection against only one marker gene. The subsequent PCR-based screens of
blasticidin-resistant clones revealed the integration of both the selected and
the unselected targeting cassettes at their original respective loci creating
complete gene disruptions. For the genes we have tested in these studies
(myosin heavy chain kinases B and C), the efficiency of the double gene
targeting event is found in the range of 2%Ð5% of all blasticidin-resistant
colonies following the transfection step. This approach for the simultaneous
disruptions of multiple genes should prove to be a valuable tool for other
laboratories interested in creating multiple gene disruptants in
Dictyostelium or other organisms where a limited number of selectable markers
are available.
Ê
Submitted by: Thomas T Egelhoff [tte@po.cwru.edu]
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Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface
of motile cells
Till Bretschneider 1, Stefan Diez 2, Kurt Anderson 2, John Heuser 3,
Margaret Clarke 4, Annette Mueller-Taubenberger 1, Jana Koehler 1 and
Guenther Gerisch 1
1 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried;
2 Max-Planck-Institut fuerÊmolekulare Zellbiologie und Genetik,
Pfotenhauer Str. 108, D-01307 Dresden;
3 Department of Cell Biology and Physiology, Washington University
ÊÊ School of Medicine,ÊSt. Louis, Missouri, USA;
4 Program in Molecular and Cell Biology, Oklahoma Medical
Research Foundation,ÊOklahoma City, Oklahoma, USA.
Ê
Current Biology, in press
Summary
Background: In the cortical region of motile cells, the actin network is
rapidly reorganized as required for movement in various directions and of
cell-to-substrate adhesion. Visualization of the fine structure and
quantitative analysis of actin network dynamics requires the combination of
high-resolution imaging with a specific fluorescent probe that highlights the
filamentous actin structures in live cells.
Results: Combining total-internal reflection fluorescence (TIRF) microscopy
with a method for labeling actin filaments in live cells, we analyze the
dynamics of actin patterns in the highly motile cells of Dictyostelium. A
rapidly restructured network of single or bundled actin filaments provides
a scaffold for the assembly of differentiated actin complexes. Two types of
these structures are characterized by recruitment of the Arp2/3 complex:
stationary foci with a lifetime of 7-10 seconds, and traveling waves. These
structures are also formed in the absence of myosin-II. Our data indicate
that Arp2/3-actin asemblies similar to those driving the protrusion of a
leading edge are freely formed at the inner face of the plasma membrane on
the bottom of the cells.
Conclusions: The actin system of highly motile cells runs far from equilibrium,
generating a multitude of patterns within a dynamic filamentous network.
Traveling waves are the most complicated patterns based on recruitment of the
Arp2/3 complex. They are governed by the propagated induction of actin
polymerization. We hypothesize that the actin system autonomously generates
primordia of specialized structures such as phagocytic cups or lamellipodia.
These primordia would represent an activated state of the actin system that
enables the cells to respond within seconds to local stimuli by chemotaxis or
phagocytic cup formation.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
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[End Dicty News, volume 21, number 17]
