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dictyNews Volume 40 Number 04
dictyNews
Electronic Edition
Volume 40, number 4
February 7, 2014
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
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Abstracts
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Both contractile axial and lateral traction force dynamics drive amoeboid
cell motility
Effie Bastounis, Ruedi Meili, Begoña Alvarez-González, Joshua Francois,
Juan C. del Álamo, Richard A. Firtel*, and Juan C. Lasheras*
(*co-senior authors). (2013)
J. Cell Biol., in press
Chemotaxing Dictyostelium cells adapt their morphology and migration
speed in response to intrinsic and extrinsic cues. Using Fourier Traction
Force Microscopy, we measured the spatiotemporal evolution of shape
and traction stresses and constructed traction tension kymographs to
analyze cell motility as a function of the dynamics of the cells’
mechanically active traction adhesions. We show that wild-type cells
migrate in a step-wise fashion mainly forming stationary traction
adhesions along their anterior-posterior axes and exerting strong
contractile axial forces. We demonstrate that lateral forces are also
important for motility, especially for migration on highly adhesive
substrates. Analysis of two mutant strains lacking distinct actin
crosslinkers (mhcA- and abp120- cells) on normal and highly adhesive
substrates supports a key role for lateral contractions in amoeboid cell
motility, while the differences in their traction adhesion dynamics
suggest these two strains use distinct mechanisms to achieve migration.
Finally, we provide evidence that the above patterns of migration may
be conserved in mammalian amoeboid cells.
Rick Firtel [rafirtel@ucsd.edu]
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Visualization of the Actin Cytoskeleton: Different F-Actin-Binding Probes
Tell Different Stories
Michael G. Lemieux, Dani Janzen, Rander Hwang, Jeannette Roldan,
Irene Jarchum, and David A. Knecht*
*Department of Molecular and Cell Biology, University of Connecticut,
Storrs, Connecticut 06269
Cytoskeleton, In Press
2013 Dec 17. doi: 10.1002/cm.21160. [Epub ahead of print]
PMID: 24347465
The actin cytoskeleton is necessary for cell viability and plays crucial roles
in cell motility, endocytosis, growth, and cytokinesis. Hence visualization
of dynamic changes in F-actin distribution in vivo is of central importance
in cell biology. This has been accomplished by the development of
fluorescent protein fusions to actin itself or to various actin-binding proteins,
actin cross-linking proteins, and their respective actin-binding domains
(ABD’s). Although these protein fusions have been shown to bind to
F-actin in vivo, we show that the fluorescent protein used for visualization
changes the subset of F-actin labeled by an F-actin ABD probe. Further,
different amino acid linkers between the fluorescent protein and ABD
induced a similar change in localization. Although different linkers and
fluorescent proteins can alter the subset of actin bound by a particular
ABD, in most cases, the fusion protein did not label all of a cell’s F-actin
all of the time. Even LimEÄcoil and GFP-actin, which have been used
extensively for cytoskeletal visualization, were highly variable in the
subsets of actin that they labeled. Lifeact, conversely, clearly labeled
cortical F-actin as well as F-actin in the anterior pseudopods of motile
cells and in macropinocytotic cups. We conclude that Lifeact most
accurately labels F-actin and is the best currently available probe for
visualization of dynamic changes in F-actin networks.
Michael Lemieux [michael.lemieux@uconn.edu]
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Linh Hai Vu, Tsuyoshi Araki, Jianbo Na, Christoph S. Clemen, Jeffrey G.
Williams and Ludwig Eichinger
PLoS ONE, accepted
Abstract
Cellular adaptation to changes in environmental osmolarity is crucial
for cell survival. In Dictyostelium, STATc is a key regulator of the
transcriptional response to hyperosmotic stress. Its phosphorylation
and consequent activation is controlled by two signaling branches,
one cGMP- and the other Ca2+-dependent, of which many signaling
components have yet to be identified. The STATc stress signalling
pathway feeds back on itself by upregulating the expression of STATc
and STATc-regulated genes. Based on microarray studies we chose
two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators
of STATc phosphorylation and generated single and double knock-out
mutants to them. Transcriptional regulation of STATc and STATc
dependent genes was disturbed in pyk3-, phg2-, and pyk3-/phg2- cells.
The absence of Pyk3 and/or Phg2 resulted in diminished or completely
abolished increased transcription of STATc dependent genes in response
to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc
levels were significantly reduced in pyk3- and phg2- cells and even further
decreased in pyk3-/phg2- cells. The reduced phosphorylation was mirrored
by a significant delay in nuclear translocation of GFP-STATc. The protein
tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits
STATc, is inhibited by stress-induced phosphorylation on S448 and S747.
Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3
is involved in the phosphorylation of PTP3 on S747. In pull-down assays
Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates
PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2-
cells. We show that Phg2 and an, as yet unknown, S448 protein kinase
are responsible for PTP3 phosphorylation and hence its inhibition, and
that Pyk3 is involved in the regulation of STATc by either directly or
indirectly activating it. Our results add further complexities to the
regulation of STATc, which presumably ensure its optimal activation
in response to different environmental cues.
Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de]
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[End dictyNews, volume 40, number 4]
