Copy Link
Add to Bookmark
Report
dictyNews Volume 18 Number 03
Dicty News
Electronic Edition
Volume 18, number 3
February 23, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
=============
Abstracts
=============
Phosphorylation Of The Myosin Regulatory Light Chain Plays A Role In
Motility and Polarity During Dictyostelium Chemotaxis
Hui Zhang(a), Deborah Wessels(a), Petra Fey(b), Karla Daniels(a),
Rex L. Chisholm(b) and David R. Soll(a)
(a)Department of Biological Sciences, University of Iowa, Iowa City,
Iowa 52242
(b)Department of Cell and Molecular Biology Northwestern University Medical
School, Chicago, IL 60611
J. Cell Sci., in press
The myosin regulatory light chain (RLC) of Dictyostelium discoideum is
phosphorylated at a single serine site in response to chemoattractant. To
investigate the role of the phosphorylation of RLC in both motility and
chemotaxis, mutants were generated in which the single phosphorylatable
serine was replaced with a nonphosphorylatable alanine. Several independent
clones expressing the mutant RLC in the RLC null mutant, mlcR-, were
obtained. These S13A mutants were subjected to high resolution computer-
assisted motion analysis to assess the basic motile behavior of cells in
the absence of a chemotatic signal, and the chemotactic responsiveness of
cells to the spatial, temporal and concentration components of natural cAMP
waves. In the absence of a cAMP signal, mutant cells formed lateral
pseudopods less frequently and crawled faster than wild type cells. In a
spatial gradient of cAMP, mutant cells chemotaxed more efficiently than
wild type cells. In the front of simulated temporal and natural waves of
cAMP, mutant cells responded normally by suppressing lateral pseudopod
formation. However, at the peak and in the back of either wave, mutant
cells did not lose cellular polarity like wild type cells. Since
depolarization at the peak and in the descending phase of the natural wave
is necessary for efficient chemotaxis, this deficiency resulted in a
decrease in the capacity of S13A mutant cells to track natural cAMP waves
relayed by wild type cells, and in the fragmentation of streams late in
mutant cell aggregation. These results reveal a regulatory pathway induced
by the peak and back of the chemotactic wave that alters RLC phosphorylation
and leads to cellular depolarization. We suggest that depolarization
requires myosin II rearrangement in the cortex facilitated by RLC
phosphorylation, which increases myosin motor function.
-----------------------------------------------------------------------------
Element analysis of the Polysphondylium pallidum gp64 promoter
Naohisa Takaoka1, Masashi Fukuzawa2, Atsushi Kato1, Tamao Saito1 and Hiroshi
Ochiai1
1. Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060-0810 Japan and 2. Wellcome Trust Building,
Department of Anatomy and Physiology, University of Dundee, Dow Street,
Dundee, DD1 4HN, UK
Biochim. Biophys. Acta, in press.
gp64 mRNA in Polysphondylium pallidum is expressed extensively during
vegetative growth, and begins to rapidly decrease at the onset of development.
To examine this unique regulation, 5 deletion analysis of the gp64 promoter
was undertaken, and two growth-phase activated elements have been found: a
food-dependent, upstream regulatory region (FUR, -222 to 170) and a
vegetatively activated, downstream region (VAD, -110 to 63). Here we
concentrate our analysis on an A1 and A2 sequences in the FUR region: A1
consists of a GATTTTTTTA sequence called a corresponding sequence and A2
consists of the direct repeat TTTGTTGTG. The cells carrying a combined
construct of A1 and A2 acted synergistically in a reporter activity. A
point mutation analysis in A1 indicates that a G residue is required for
the activation of A1. From analyses of promoter regulation in a liquid or
a solid medium, the promoter activity of the cells fed on bacteria in
A-medium (axenic medium for Polysphondylium) or grown in A-medium alone
was only one-fourth of that of the cells fed on bacteria.
-----------------------------------------------------------------------------
Quantitation of the distribution and flux of myosin-II during cytokinesis.
Douglas N. Robinson#, Guy Cavet*, Hans M. Warrick and James A. Spudich
Departments of Biochemistry and Developmental Biology, Stanford University
School of Medicine, Stanford, CA 94305-5307
Email: Douglas.Robinson@jhu.edu; jspudich@cmgm.stanford.edu
#Current address: Department of Cell Biology, Johns Hopkins University School
of Medicine, 725 N. Wolfe St., Baltimore, MD 21205
*Current address: Rosetta Inpharmatics, 12040 115th Ave NE, Kirkland,
WA 98034.
These two authors contributed equally to this work.
BMC Cell Biology 3:4, 2002.
Background: During cytokinesis, the cell's equator contracts against the
cell's global stiffness. Identifying the biochemical basis for these
mechanical parameters is essential for understanding how cells divide. To
achieve this goal, the distribution and flux of the cell division machinery
must be quantified. Here we report the first quantitative analysis of the
distribution and flux of myosin-II, an essential element of the contractile
ring.
Results: The fluxes of myosin-II in the furrow cortex, the polar cortex,
and the cytoplasm were examined using ratio imaging of GFP fusion proteins
expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the
furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold
higher than in the cytoplasm. The myosin-II in the furrow cortex, however,
represents only 10% of the total cellular myosin-II. An estimate of the
minimal amount of this motor needed to produce the required force for cell
cleavage fits well with this 10% value. The cell may, therefore, regulate
the amount of myosin-II sent to the furrow cortex in accordance with the
amount needed there. Quantitation of the distribution and flux of a mutant
myosin-II that is defective in phosphorylation-dependent thick filament
disassembly confirms that heavy chain phosphorylation regulates normal
recruitment to the furrow cortex.
Conclusion: The analysis indicates that myosin-II flux through the cleavage
furrow cortex is regulated by thick filament phosphorylation. Further, the
amount of myosin-II observed in the furrow cortex is in close agreement with
the amount predicted to be required from a simple theoretical analysis.
-----------------------------------------------------------------------------
Outside-In Signaling of Cellulose Synthesis by a Spore Coat Protein in
Dictyostelium
Christopher M. West, Ping Zhang, Aiko C. McGlynn and Lee Kaplan
Dept. of Anatomy and Cell Biology, College of Medicine, University of
Florida, Gainesville, FL 32610-0235 USA
Eukaryotic Cell, in press.
ABSTRACT
The spore coat of Dictyostelium is formed de novo from proteins secreted
from vesicles and cellulose synthesized across the plasma membrane as
differentiating spores rise up the stalk. The mechanism by which these
events are coordinated is not understood. In the course of experiments
designed to test the function of the inner layer coat protein SP85 (PsB),
expression of a specific partial length fragment was found to interrupt
coat assembly after protein secretion and prior to cellulose synthesis in
85% of the cells. This fragment consisted of SP85's N-terminal domain,
containing prespore vesicle targeting information, and its Cys-rich
C1-domain. The effect of the NC1 fusion was non-cell autonomous in
interstrain chimeras, suggesting it acted at the cell surface. SP85-null
spores presented an opposite phenotype in which spores differentiated
prematurely before reaching the top of the stalk, and cellulose was
slightly overproduced in a disorganized fashion. A similar though less
severe phenotype occurred when a fusion of the N- and C2-domains was
expressed. In a double mutant, absence of SP85 was epistatic to NC1-
expression suggesting that NC1 inhibited SP85 function. Together, these
results suggest the existence of an outside-in signaling pathway that
constitutes a checkpoint to ensure that cellulose synthesis does not occur
until coat proteins are properly organized at the cell surface and stalk
formation is complete. Checkpoint execution is proposed to be regulated by
SP85, which is in turn under the influence of contacts with other coat
proteins that are competed by the presence of NC1 and NC2.
-----------------------------------------------------------------------------
Ralph Grf
Adolf-Butenandt-Institut / Zellbiologie, Universitt Mnchen, Schillerstr.
42, D-80336 Mnchen, Germany
J. Cell Sci. in press
Dictyostelium Nek2 (DdNek2) is the first structural and functional non-
vertebrate homologue of human Nek2, a NIMA-related serine/threonine kinase
required for centrosome splitting in early mitosis. DdNek2 shares 43%
overall amino-acid identity with its human counterpart and even 54% within
the catalytic domain. Both proteins can be subdivided in an N-terminal
catalytic domain, a leucine zipper, and a C-terminal domain. Kinase assays
with bacterially expressed DdNek2 and C-terminal deletion mutants revealed
that catalytic activity requires the presence of the leucine zipper and that
autophosphorylation occurs at the C-terminus. Microscopic analyses with
DdNek2 antibodies and expression of a GFP-DdNek2 fusion protein in
Dictyostelium showed that DdNek2 is a permanent centrosomal resident and
suggested that it is a component of the centrosome core. The GFP-DdNek2
overexpressing mutants frequently exhibit supernumerary microtubule-
organizing centers (MTOCs). This phenotype did not require catalytic
activity because it was also observed in cells expressing inactive GFP-K33R.
However, it was caused by overexpression of the C-terminal domain since it
also occurred in GFP-mutants expressing only the C-terminus or a leucine
zipper/C-terminus construct but not in those mutants expressing only the
catalytic domain or a catalytic domain/leucine zipper construct. These
results suggest that DdNek2 is involved in the formation of MTOCs.
Furthermore, the localization of the GFP-fusion proteins revealed two
independent centrosomal targeting domains of DdNek2, one within the
catalytic or leucine zipper domain and one in the C-terminal domain.
-----------------------------------------------------------------------------
Liu, T., Mirschberger, C., Chooback, L., Arana, Q., Dal Sacco, Z.,
MacWilliams, H., and Clarke, M.
Altered expression of the 100-kDa subunit of the Dictyostelium vacuolar
proton pump impairs enzyme assembly, endocytic function, and cytosolic pH
regulation.
J. Cell Sci., in press.
The vacuolar proton pump (V-ATPase) appears to be essential for viability of
Dictyostelium cells. To investigate the function of VatM, the 100-kDa
transmembrane V-ATPase subunit, we altered its level. By means of homologous
recombination, the promoter for the chromosomal vatM gene was replaced with
the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6
promoter is much more active in cells growing axenically than on bacteria.
Thus, transformants were selected under axenic growth conditions, then
shifted to bacteria to determine the consequences of reduced vatM expression.
When VatMpr cells were grown on bacteria, the level of the 100-kDa V-ATPase
subunit dropped, cell growth slowed, and the A subunit, a component of the
peripheral catalytic domain of the V-ATPase, became mislocalized. These
defects were complemented by transformation of the mutant cells with a
plasmid expressing vatM under the control of its own promoter. Although the
principal locus of vacuolar proton pumps in Dictyostelium is membranes of
the contractile vacuole system, mutant cells did not manifest osmoregulatory
defects. However, bacterially-grown VatMpr cells did exhibit substantially
reduced rates of phagocytosis and a prolonged endosomal transit time. In
addition, mutant cells manifested alterations in the dynamic regulation of
cytosolic pH that are characteristic of normal cells grown in acid media,
suggesting that the V-ATPase also plays a role in cytosolic pH regulation.
-----------------------------------------------------------------------------
Transfer RNA gene-targeted retrotransposition of Dictyostelium TRE5-A
into a chromosomal UMP synthase gene trap
Peter Beck, Theodor Dingermann and Thomas Winckler
Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M.
(Biozentrum), Frankfurt am Main, Germany
J. Mol. Biol., in press
The genome of the eukaryotic microorganism Dictyostelium discoideum
hosts a family of seven non-long terminal repeat retrotransposons (TREs)
which show remarkable insertion preferences near tRNA genes. We
developed an in vivo assay to detect tRNA gene-targeted
retrotransposition of endogenous TREs in a reporter strain of D.
discoideum. A tRNA gene positioned within an artificial intron was
placed into the D. discoideum UMP synthase gene. This construct was
inserted into the D. discoideum genome and presented as a landmark for
de novo TRE insertions. We show that the tRNA gene-tagged UMP synthase
gene was frequently disrupted by de novo insertions of endogenous TRE5-A
copies, thus rendering the resulting mutants resistent to 5-fluoro
orotic acid selection. Approx. 96% of all isolated 5-FOA-resistent
clones contained TRE5-A insertions, whereas the remaining 4% resulted
from transposition-independent mutations. The inserted TRE5-As showed
complex structural variations and were found about 50 bp upstream of the
reporter tRNA gene similar to previously analysed genomic copies of
TRE5-A. No integration by other members of the TRE family was observed.
We found that only 51% of the de novo insertions were derived from
autonomous TRE5-A.1 copies. The remaining 49% of new insertions were due
to TRE5-A.2 elements, which lack the proteins required for reverse
transcription and integration, but retained functional promoter sequences.
-----------------------------------------------------------------------------
[End Dicty News, volume 18, number 3]
