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dictyNews Volume 23 Number 20
Dicty News
Electronic Edition
Volume 23, number 20
December 31, 2004
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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Loss-of-Function Mutations Identified in the Helical Domain of the G
Protein alpha- Subunit, Galpha2, of Dictyostelium discoideum.
Robert E. Gundersen, Jianxin You, Steven Rauch, Kate Farnham, Christopher
McCarty, Nicholas Willis and Alison Prince
Biochemistry, Microbiology and Molecular Biology, University of Maine
accepted: BBA - General Subjects
The guanine nucleotide binding regulatory proteins (G proteins) play
essential roles in a wide variety of physiological processes such as,
vision, hormone responses, olfaction, immune response, and development.
The heterotrimeric G proteins, consists of an alpha, beta and gamma-
subunits and act as molecular switches to relay information from
transmembrane receptors to intracellular effectors. The switch mechanism
is a function of the inherent GTPase activity of the alpha-subunit. The
alpha-subunit is comprised of two domains, the GTPase domain and the
Helical domain. The GTPase domain performs all of the known alpha-
subunit functions while little is know about the role of the Helical
domain. To gain a better understanding of alpha-subunit function, we
performed a screen for loss-of-function mutations, using the Galpha2-
subunit of Dictyostelium. Galpha2 is essential for the developmental life
cycle of Dictyostelium. It is known that loss of Galpha2 function results
in a failure of cells to enter the developmental phase, producing a
visibly abnormal phenotype. This allows easy identification of amino
acids essential to Galpha2 function. A library of random point mutations
in the galpha2 cDNA was constructed using low fidelity Polymerase Chain
Reaction (PCR). The library was then expressed in a galpha2 null cell
line and screened for loss-of-function mutations. Mutations were
identified in isolated clones by sequencing the galpha2 insert. To date
sixteen, single amino acids changes have been identified in Galpha2 which
result in loss-of-function. Of particular interest are seven mutations
found in the Helical domain of the alpha-subunit. These loss-of-function
mutations in the alpha-subunit Helical domain may provide important
insight into its function.
Submitted by: Robert Gundersen [gundersn@maine.edu]
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An auto-regulatory circuit for long-range self-organization in
Dictyostelium cell populations
Satoshi Sawai, Peter A. Thomason & Edward C. Cox
Department of Molecular Biology, Princeton University, Princeton, New
Jersey 08544, USA
Nature, in press
Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular
structure by chemotaxis, moving towards propagating waves of cyclic AMP
that are relayed from cell to cell. Organizing centres are not formed by
founder cells, but are dynamic entities consisting of cores of outwardly
rotating spiral waves that self-organize in a homogeneous cell population.
Spiral waves are ubiquitously observed in chemical reactions as well as in
biological systems. Although feedback control of spiral waves in spatially
extended chemical reactions has been demonstrated in recent years, the
mechanism by which control is achieved in living systems is unknown. Here
we show that mutants of the cyclic AMP/protein kinase A pathway show
periodic signalling, but fail to organize coherent long-range wave
territories, owing to the appearance of numerous spiral cores. A
theoretical model suggests that autoregulation of cell excitability
mediated by protein kinase A acts to optimize the number of signalling
centres.
Submitted by: Satoshi Sawai [ssawai@molbio.princeton.edu]
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Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-
independent processes in Dictyostelium
Shi Shu, Xiong Liu and Edward D. Korn*
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute,
Bethesda, Maryland 20892
Proc. Natl. Acad. Sci., in press
Blebbistatin, a cell-permeable inhibitor of class-II myosins, was
developed to provide a tool for studying the biologic roles of myosin II.
Consistent with this, we find that blebbistatin inhibits three myosin II-
dependent processes in Dictyostelium: growth in suspension culture,
capping of concanavalin A receptors and development to fruiting bodies and
does not inhibit growth on plates, which does not require myosin II. As
expected, macropinocytosis (myosin I-dependent), contractile vacuole
activity (myosin V-dependent) and phagocytosis (myosin VII- dependent),
none of which requires myosin II, are not inhibited by blebbistatin in
myosin II-null cells but, unexpectedly, blebbistatin does inhibit
macropinocytosis and phagocytosis by cells expressing myosin II.
Expression of catalytically inactive myosin II in myosin II-null cells
also inhibits macropinocytosis and phagocytosis. Both blebbistatin-
inhibited myosin II and catalytically inactive myosin II form cytoplasmic
aggregates, which may be why they inhibit myosin II-independent
processes, but neither affects the distribution of actin filaments in
vegetative cells or actin and myosin distribution in dividing or polarized
cells. Blebbistatin also inhibits cell streaming and plaque expansion in
myosin II-null cells. Our results are consistent with myosin II being the
only Dictyostelium myosin that is inhibited by blebbistatin but also show
that blebbistatin-inactivated myosin II inhibits some myosin II-
independent processes and that blebbistatin inhibits other activities in
the absence of myosin II.
Submitted by: Edward D. Korn [edk@nih.gov]
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[End Dicty News, volume 23, number 20] 
