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dictyNews Volume 38 Number 18
dictyNews
Electronic Edition
Volume 38, number 18
July 20, 2012
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.
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Abstracts
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Myosin Heavy Chain Kinases Play Essential Roles In Ca2+, But Not cAMP,
Chemotaxis And The Natural Aggregation of Dictyostelium discoideum
Deborah Wessels, Daniel F. Lusche, Paul A. Steimle, Amanda Scherer,
Spencer Kuhl, Kristen Wood, Brett Hanson, Thomas T. Egelhoff and
David R. Soll
Journal of Cell Science, in press
Behavioral analyses of the deletion mutants of the four known myosin II
heavy chain (Mhc) kinases of D. discoideum revealed that all played a
minor role in the efficiency of basic cell motility, but none played a role in
chemotaxis in a spatial gradient of cAMP generated in vitro. However,
each of the two kinases MhckA and MhckC, was essential for chemotaxis
in a spatial gradient of Ca2+, shear induced directed movement, and
reorientation in the front of waves of cAMP during natural aggregation.
The mutant phenotypes of mhckA- and mhckC- were highly similar to that
of the Ca2+ channel/receptor mutant iplA- and the myosin II phosphorylation
mutant 3XALA, which produces constitutively unphosphorylated myosin II.
These results demonstrate that IplA, MhckA and MhckC play a selective
role in chemotaxis in a spatial gradient of Ca2+, but not cAMP and suggest
that Ca2+ chemotaxis plays a role in the orientation of cells in the front of
cAMP waves during natural aggregation.
Submitted by Deborah Wessels [deborah-wessels@uiowa.edu]
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The balance in the delivery of ER components and the vacuolar proton
pump to the phagosome depends on myosin IK in Dictyostelium.
Regis Dieckmann1#, Aurelie Gueho1, Roger Monroy1, Thomas Ruppert2,
Gareth Bloomfield3 and Thierry Soldati1*
Molecular & Cellular Proteomics, in press
1Department de Biochimie, Faculte des Sciences, University de Geneve,
Sciences II, 30 quay Ernest Ansermet, CH-1211 Geneve-4, Switzerland
2Core Facility for Mass Spectrometry and Proteomics, Zentrum fuer
Molekulare Biologie der Universitaet Heidelberg (ZMBH), Im Neuenheimer
Feld 282, D-69120 Heidelberg, Germany
3MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
#Present address: : Klinisches Institut fuer Pathologie, 1090 Wien, Austria
*Corresponding author
Molecular & Cellular Proteomics, in press
In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1)
and the class I myosin MyoK directly interact and couple actin dynamics to
membrane deformation during phagocytosis. Together with the kinase PakB,
they build a regulatory switch that controls the efficiency of uptake of large
particles. As a basis for further functional dissection, exhaustive phagosome
proteomics was performed and established that about 1300 proteins participate
in phagosome biogenesis. Then, quantitative and comparative proteomic analysis
of phagosome maturation was performed to investigate the impact of the absence
of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis
(2D-DIGE) of phagosomes isolated from myoK-null and abp1-null cells were used
to determine the relative abundance of proteins during the course of maturation.
Immunoblot profiling showed that absence of Abp1 alters the maturation profile of
its direct binding partners such as actin and the Arp2/3 complex, suggesting that
Abp1 directly regulates actin dynamics at the phagosome. Comparative 2D-DIGE
analysis resulted in the quantification of mutant-to-wild type abundance ratios at
all stages of maturation for over one hundred identified proteins. Coordinated
temporal changes in these ratio profiles determined the classification of identified
proteins into functional groups. Ratio profiling revealed that the early delivery of
ER proteins to the phagosome was affected by the absence of MyoK and was
coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump
and Rab11 GTPases. As direct functional consequences, a delayed acidification
and a reduced intra-phagosomal proteolysis were demonstrated in vivo in
myoK-null cells. In conclusion, the absence of MyoK alters the balance of the
contributions of the ER and an endo-lysosomal compartment, and slows down
phagosome acidification as well as the speed and efficiency of particle
degradation inside the phagosome.
Submitted by Thierry Soldati [thierry.soldati@unige.ch]
-------------------------------------------------------------------------------------
Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has
effects on cell migration.
Jessica S Kelsey, Nathan M Fastman, Elizabeth F Noratel and Daphne D
Blumberg*
Molecular Biology of the Cell, In Press
The ampA gene has a role in cell migration in Dictyostelium discoideum.
Cells overexpressing AmpA show an increase in cell migration, forming
large plaques on bacterial lawns. A second site suppressor of this
ampA-overexpressing phenotype identified a previously uncharacterized
gene, ndm which is described here. The Ndm protein is predicted to
contain a coiled-coil BAR like domain, a domain involved in endocytosis
and membrane bending. Ndm knockout and Ndm-mRFP expressing cell lines
were used to establish a role for ndm in suppressing endocytosis. An
increase in the rate of endocytosis and in the number of endosomes was
detected in ndm- cells. During migration ndm- cells formed numerous
endocytic cups instead of the broad lamellipodia structure characteristic
of moving cells. A second lamellipodia based function, cell spreading, was
also defective in the ndm- cells. The increase in endocytosis and the
defect in lamellipodia formation were associated with reduced chemotaxis
in ndm- cells. Immunofluorescence results and GST pull down assays
revealed an association of Ndm with coronin and F-actin. The results
establish Ndm as a gene important in regulating the balance between
formation of endocytic cups and lamellipodia structures.
Submitted by Daphne Blumberg [blumberg@umbc.edu]
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[End dictyNews, volume 38, number 18]
