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dictyNews Volume 26 Number 06
dictyNews
Electronic Edition
Volume 26, number 6
February 24, 2006
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
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Dabp1 regulates pseudopodium number in chemotaxing Dictyostelium cells
Yanqin Wang and Theresa J. O'Halloran
Journal of Cell Science, in press
In Dictyostelium, chemotactic aggregation is a dynamic pathway in which
cells respond to extracellular signals, polarize, and move with strong
persistence into aggregation centers. Actin and actin-associated proteins
play an important role in controlling the morphological changes and the
directed movement of cells during chemotactic aggregation. Recently we
identified a homologue of Abp1 in Dictyostelium (Dabp1). The first actin
binding protein identified in yeast, abp1 plays a key role in actin-based
endocytosis in these cells. More recently, a role in receptor-mediated
endocytosis has been identified for Abp1 in mammalian cells. To explore
possible functions of Dabp1 in Dictyostelium, we examined the phenotypes
of cells that overexpressed the Dabp1 protein and cells that eliminated
Dabp1 expression. In these mutants most actin-based processes were intact.
However, cell motility was altered during early development. During
chemotactic streaming up to 25% (+4%) of Dabp1 null cells had multiple
pseudopods while 92% wildtype cells had a single leading edge and a single
uropod. Similarly, about 90% of cells that overexpressed Dabp1 projected
multiple pseudopodia during chemotactic streaming, and displayed reduced
rates of cell movement. Expression of the SH3 domain of Dabp1 showed this
domain to be an important determinant in regulating pseudopod number.
These results suggest that Dabp1 controls pseudopod number and motility
in early stages of chemotactic aggregation in Dictyostelium.
Submitted by: Terry OÕHalloran [t.ohalloran@mail.utexas.edu]
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Calmodulin-Mediated Signaling in Dictyostelium discoideum: CaMBOT Isolation
and Characterization of the Novel Poly-Domain Protein Nucleomorphin and
Other Calmodulin Binding Proteins.
Danton H. O'Day, Department of Biology, University of Toronto at Mississauga,
Mississauga, Ontario L5L 1C6 CANADA
Chapter 6, In: "Focus on Cellular Signalling Research", Nova Science
Publishers, Inc. (in press)
Calmodulin (CaM) is an essential protein in the model eukaryote Dictyostelium
discoideum where it mediates numerous events including chemotaxis,
gametogenesis, fertilization, and spore germination. Profiling using the
CaM-binding overlay technique (CaMBOT) has revealed that well over four-dozen
calcium-dependent and -independent CaM-binding proteins (CaMBPs) are present
in Dictyostelium, some of which are linked to these CaM-mediated processes.
Individual CaMBPs have been localized to specific sub-cellular locales and to
show varying patterns of developmental expression. CaMBOT also is used to
isolate cDNAs encoding CaMBPs from an expression library from mid-to-late
multicellular development of Dictyostelium. Previously studied CaMBPs such as
calcineurin A (CNA) and regulatory myosin light chain (RMLC) were identified
in this way. Novel proteins were also identified. Nucleomorphin is a novel
nuclear CaMBP, expressed as two main isoforms NumA1 and 2, which localizes to
the nucleoplasmic periphery. NumA has at least one bipartite NLS
(48KKSYQDPEIIAHSRPRK66) and a single CaM-binding domain
(171EDVSRFIKGKLLQKQQKIYKDLERF195) that interacts with CaM in both a
Ca2+-dependent and Ca2+-independent manner. NumA1 contains an extensive
glu/asp or DEED repeat that regulates nuclear number. Yeast two hybrid and
co-immunoprecipitation studies have identified calcium binding protein CBP4a
as an interacting protein that binds to the DEED repeat. NumA2 also has a
breast cancer C-terminus (BRCT) domain, two poly-N tracts plus a second,
putative CaMBD. Proteins not previously known to be CaMBPs were also
identified by CaMBOT probing of the Dictyostelium developmental expression
library, including phosphoglycerate kinase, thymidine kinase and histone H1.
Dictyostelium phosphoglycerate kinase (DdPGK) binds CaM in a Ca2+-dependent
manner with CaM negatively regulating its activity in vitro. The identified
CaMBD shows 80% identity with PGKs from diverse organisms and is localized
adjacent to mutation sites underlying certain human diseases. Thymidine
kinase (DdTK1) is a Ca2+-dependent CaMBP and its in vitro activity is
slightly enhanced by CaM and inhibited by CaM antagonists. Histone H1 (DdH1)
was isolated as a CaMBP and binds CaM in vitro but its mode of CaM-binding
remains elusive. Each of the CaMBPs we have identified is discussed in terms
of its individual roles as well as its potential interaction with other
CaMBPs and other proteins in regulating specific CaM-dependent cellular
processes including the cell cycle.
Submitted by: Dan OÕDay [doday@utm.utoronto.ca]
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The contribution of the endoplasmic reticulum to Ca2+ signals in
Dictyostelium depends on extracellular Ca2+
Paul R. Fisher* and Zofia Wilczynska.
Department of Microbiology, La Trobe University, Victoria 3086, Australia.
FEMS Microbiology Letters, in press
We recently reported the first molecular genetic evidence that Dictyostelium
Ca2+ responses to chemoattractants include a contribution from the
endoplasmic reticulum (ER) - responses are enhanced in mutants lacking
calreticulin or calnexin, two major Ca2+-binding proteins in the ER, even
though the influx of Ca2+ into the mutants is reduced. Compared to wild type
cells, the ER in the mutants contributes at least 30-70 nM additional Ca2+
to the responses. Here we report that this additional ER contribution to the
cytosolic Ca2+ signal depends upon extracellular Ca2+ - it does not occur in
the absence of extracellular Ca2+, increases to a maximum as the
extracellular Ca2+ levels rise to 10 uM and then remains constant at
extracellular Ca2+ concentrations up to at least 250 uM. These results
suggest that Ca2+ influx causes the intracellular release, in the simplest
scenario by a mechanism involving Ca2+-induced Ca2+ release from the ER. By
way of contrast, we show that Ca2+ responses to mechanical stimulation are
reduced, but still occur in the absence of extracellular Ca2+. Unlike the
responses to chemoattractants, mechanoresponses thus include contributions
from the ER that are independent of extracellular Ca2+.
Submitted by: Paul Fisher [fisher@lumi.micro.latrobe.edu.au]
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[End dictyNews, volume 26, number 6] 
