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dictyNews Volume 24 Number 10
Dicty News
Electronic Edition
Volume 24, number 10
April 15, 2005
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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Subsecond reorganization of the actin network in cell motility and chemotaxis
Stefan Diez 1, Guenther Gerisch 2, Kurt Anderson 1, Annette
Mueller-Taubenberger 2 and Till Bretschneider 2
1 Max-Planck-Institut fuer molekulare Zellbiologie und Genetik,
D-01307 Dresden, Germany
2 Max-Planck-Institut fuer Biochemie,
D-82152 Martinsried, Germany
Proc. Natl. Acad. Sci. USA, in press
Actin networks are continuously reorganized in cells that rapidly change
their shape. Applying total internal reflection fluorescence (TIRF) microscopy
at acquisition rates of 10 to 20 Hz, we measured an average growth rate of
3 mm x sec-1 for filamentous actin structures throughout the entire
substrate-attached cortex of Dictyostelium cells. New filaments often proceed
along pre-existing ones, resulting in bundle formation concurrent with
filament growth. In cells that orientate in a gradient of chemoattractant,
prominent assemblies of actin enriched in the Arp2/3 complex are inserted into
the network, primarily at the base of filopods that point into the direction
of the gradient. We propose that high turnover rates of actin filaments confer
the plasticity to the cell cortex that is required for rapid accommodation to
external stimuli.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
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A cysteine-rich extracellular protein containing a PA14 domain mediates
quorum sensing in Dictyostelium
Alexandra Kolbinger1, Tong Gao2, Debbie Brock2, Robin Ammann2 Axel Kisters1,
Joseph Kellermann3, Diane Hatton2, Richard H. Gomer2*, and Birgit Wetterauer1,
1 Zoologisches Institut der Ludwig-Maximilians-Universitt, Munich, Germany
2 Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Biology, Rice University, Houston, Texas, USA
3 Max-Planck-Institut fr Biochemie, Martinsried, Germany
Eukaryotic Cell, in press
Much remains to be understood about quorum sensing factors that allow cells
to sense their local density. Dictyostelium discoideum is a simple eukaryote
that grows as single-celled amoebae and switches to multicellular development
when food becomes limited. As the growing cells reach a high density, they
begin expressing discoidin genes. The cells secrete an unknown factor, and
at high cell densities the concomitant high levels of the factor induce
discoidin expression. We report here the enrichment of Discoidin Inducing
Complex (DIC), an ~400 kDa protein complex that induces discoidin expression
during growth and development. Two proteins in the DIC preparation, DicA1
and DicB, were identified by sequencing of proteolytic digests. DicA1 and
DicB were expressed in E. coli and tested for their ability to induce
discoidin during growth and development. Recombinant DicB was unable to
induce discoidin expression while recombinant DicA1 was able to induce
discoidin expression. This suggests that DicA1 is an active component of
DIC and indicates that posttranslational modification is dispensable for
activity. DicA1 mRNA is expressed in vegetative and developing cells.
The mature secreted form of DicA1 is 80 kDa, and has a 24 amino acid
cysteine rich repeat that is similar to repeats in Dictyostelium proteins
such as the extracellular matrix protein ecmB/PstA, the prespore cell
inducing factor PSI, and the cAMP phosphodiesterase inhibitor PDI.
Together, the data suggest that DicA1 is a component of a secreted quorum
sensing signal regulating discoidin gene expression during Dictyostelium
growth and development.
Submitted by: Richard Gomer [richard@rice.edu]
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Isolation and characterization of Dictyostelium thymidine kinase 1 as a
calmodulin binding protein
Danton H. OâDay, Munmun Chatterjee-Chakraborty, Stephanie Wagler and
Michael A. Myre
Department of Biology, University of Toronto at Mississauga, Mississauga,
ON. Canada
Biochemical Biophysical Research Communications, in press
Probing of a cDNA expression library from multicellular development of
Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe
(35S-VU1-CaM) led to the isolation of a cDNA encoding a putative
CaM-binding protein (CaMBP). The cDNA contained an open reading frame of
951bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led
to highly significant matches with cytosolic thymidine kinases (TK1;
EC 2.7.1.21) from a diverse number of species including humans (7e-56;
59% Identities; 75% Positives) indicating that the encoded protein is
D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized
in this organism. In keeping with its sequence similarity with DdTK1,
antibodies against humanTK1 recognize DdTK1, which is expressed during
growth but decreases in amount after starvation. A CaM-binding domain
(CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus
two constructs (DdTKdeltaC36, DdTKdeltaC75) possessing the domain were
shown to bind CaM in vitro but only in the presence of calcium while a
construct (DdTKdeltaN72) lacking the region failed to bind to CaM. Thus
DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from
vertebrates to viruses show that CaM-binding region is highly conserved.
The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting
CaM might affect the activity of this kinase. Recombinant DdTK is
enzymatically active and showed stimulation by CaM (113 ± 0.5%) an in vitro
enhancement that was prevented by co-addition of the CaM antagonists W7
(91.2 ± 0.8%) and W13 (96.6 ± 0.6%). The discovery that TK1 from
D. discoideum, and possibly other species including humans and a large
number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues
for research on this medically relevant protein.
Submitted by: Danton H. O'Day [doday@utm.utoronto.ca]
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An N-terminal nuclear localization sequence but not the calmodulin-binding
domain mediates nuclear localization of nucleomorphin, a protein that
regulates nuclear number in Dictyostelium
Michael A. Myre and Danton H. OâDay
Department of Biology, University of Toronto at Mississauga, Mississauga,
ON. Canada
Biochemical Biophysical Research Communications, in press
Nucleomorphin is a novel nuclear calmodulin (CaM) binding protein (CaMBP)
containing an extensive DEED (glu/asp repeat) domain that regulates
nuclear number. GFP-constructs of the 38kDa NumA1 isoform localize as
intranuclear patches adjacent to the inner nuclear membrane. The
translocation of CaMBPs into nuclei has previously been shown by others
to be mediated by both classic nuclear localization sequences (NLSs) and
CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD
(171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent
binding motifs and an IQ-like motif for calciumöindependent binding. GFP
constructs containing only NumA1 residues 1-129, lacking the DEED and
CaMBDs, still localized as patches at the internal periphery of nuclei
thus ruling out a direct role for the CaMBD in nuclear import. These
constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that
includes both a putative bipartite and classical NLS. GFP-bipartite NLS
constructs localized uniformly within nuclei but not as patches. As with
previous work, removal of the DEED domain resulted in highly multinucleate
cells. However as shown here, multinuclearity only occurred when the NLS
was present allowing the protein to enter nuclei. Site-directed mutation
analysis in which the NLS was changed to 48EF49 abolished the stability of
the GFP fusion at the protein but not RNA level preventing subcellular
analyses. Cells transfected with the 48EF49 construct exhibited slowed
growth when compared to parental AX3 cells and other GFP-NumA1 deletion
mutants. In addition to identifying an NLS that is sufficient for nuclear
translocation of nucleomorphin and ruling out CaM-binding in this event,
this work shows that the nuclear localization of NumA1 is crucial to its
ability to regulate nuclear number in Dictyostelium.
Submitted by: Danton H. O'Day [doday@utm.utoronto.ca]
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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. OâDay
Department of Biology, University of Toronto at Mississauga, Mississauga,
Ontario L5L 1C6, CANADA
In: Progress in Cellular Signalling Research, Nova Science Publishers,
Inc. New York, 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: Danton H. O'Day [doday@utm.utoronto.ca]
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[End Dicty News, volume 24, number 10] 
