dictyNews
Electronic Edition
Volume 40, number 4
February 7, 2014

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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 [[log in to unmask]]
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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[End dictyNews, volume 40, number 4]