dictyNews
Electronic Edition
Volume 32, number 12
May 1, 2009

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Abstracts
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Identification and cell cycle-dependent localization of nine
novel, genuine centrosomal components in Dictyostelium
discoideum

Irene Schulz, Alexander Erle, Ralph Gräf, Anne Krüger, Heiner Lohmeier,
SaschaPutzler, Matthias Samereier, Sebastian Weidenthaler
University of Potsdam, Institute for Biochemistry and Biology, Dept. of
Cell BiologyKarl-Liebknecht-Strasse 24-25, Haus 26
14476 Potsdam-Golm, Germany


Cell Motility and the Cytoskeleton: Mechanics and Dynamics of the  
Cytoskeleton

The centrosome is the main microtubule-organizing center and constitutes
the largest protein complex in a eukaryotic cell. The Dictyostelium  
centrosome
is an established model for acentriolar centrosomes and it consists of  
a layered
core structure surrounded by a so-called corona, which harbors  
microtubule
nucleation complexes. We have identified 34 new centrosomal candidate  
proteins
through mass spectrometrical analysis of the proteome of isolated  
Dictyostelium
centrosomes. Here we present a characterization of 12 centrosomal  
candidate
proteins all featuring coiled coil regions and low expression levels,  
which are the
most common attributes of centrosomal proteins. We used GFP fusion  
proteins
to localize the candidate proteins in whole cells and on microtubule- 
free, isolated
centrosomes. Thus we were able to identify nine new genuine  
centrosomal proteins
including a putative orthologue of Cep192, an interaction partner of  
polo-like
kinase 4 in human centriole biogenesis. In this respect, centrosomal  
localization
of the only polo-like kinase in Dictyostelium, Plk, is also shown in  
this work.
Using confocal deconvolution microscopy, four components, CP39,CP55,  
CP75
and CP91 could be clearly assigned to the so far almost uncharacterized
centrosomal core structure, while CP148 and Cep192 localized to a zone
between that of corona marker and core proteins. Finally, CP103 and  
CP248
were constituents of the corona. In contrast, NE81 was localized at  
the nuclear
envelope and three others, an orthologue of the spindle checkpoint  
component
Mad1, the novel Cenp68, and the centrosomal CP248 were observed at the
centromeres, which are clustered and linked to the centrosome throughout
the entire cell cycle.


Submitted by: Irene Schulz [[log in to unmask]]
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The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence
of External Cues.

Leonard Bosgraaf, and Peter J.M. Van Haastert


PLOS ONE, in press

Eukaryotic cells extend pseudopodia for movement. In the absence of
external cues, cells move in random directions, but with a strong  
element
of persistence that keeps them moving in the same direction Persistence
allows cells to disperse over larger areas and is instrumental to enter
new environments where spatial cues can lead the cell. Here we explore
cell movement by analyzing the direction, size and timing of ~2000
pseudopodia that are extended by Dictyostelium cells. The results show
that pseudpopod are extended perpendicular to the surface curvature at
the place where they emerge. The location of new pseudopods is not  
random
but highly ordered. Two types of pseudopodia may be formed: frequent  
splitting
of an existing pseudopod, or the occasional extension of a de novo  
pseudopod
at regions devoid of recent pseudopod activity. Split-pseudopodia are  
extended
at ~60 degrees relative to the previous pseudopod, mostly as alternating
Right/Left/Right steps leading to relatively straight zigzag runs. De  
novo
pseudopodia are extended in nearly random directions thereby  
interrupting
the zigzag runs. Persistence of cell movement is based on the ratio of  
split
versus de novo pseudopodia. We identify PLA2 and cGMP signaling
pathways that modulate this ratio of splitting and de novo pseudopodia,
and thereby regulate the dispersal of cells. The observed ordered
extension of pseudopodia in the absence of external cues provides a
fundamental insight into the coordinated movement of cells, and might
form the basis for movement that is directed by internal or external  
cues.


Submitted by: Peter Van Haastert [[log in to unmask]]
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Regulation of the formation and trafficking of vesicles from Golgi by  
PCH
Family Proteins During Chemotaxis

S. Lee, J. W. Han#, L. Leeper, J. S. Gruver, C. Y. Chung*

Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, TN 37232-6600


BBA-Molecular Cell Research, In press

Previous study demonstrated that WASP localizes on vesicles during
Dictyostelium chemotaxis and these vesicles appear to be preferentially
distributed at the leading and trailing edge of migrating cells.  In  
this
study, we have examined the role of PCH family proteins, Nwk/Bzz1p-like
protein (NLP) and Syndapin-like protein (SLP), in the regulation of the
formation and trafficking of WASP-vesicles during chemotaxis. NLP and  
SLP
appear to be functionally redundant and deletion of both nlp and slp  
genes
cause the loss of polararized F-actin organization and significant  
defects
in chemotaxis. WASP and NLP are colocalized on vesicles and interactions
between two molecules via the SH3 domain of NLP/SLP and the proline-rich
repeats of WASP are required for vesicle formation from Golgi.  
Microtubules
are required for polarized trafficking of these vesicles as vesicles  
showing
high directed mobility are absent in cells treated with nocodazole. Our
results suggest that interaction of WASP with NLP/SLP is required for  
the
formation and trafficking of vesicles from Golgi to the membrane, which
might play a central role in the establishment of cell polarity during
chemotaxis.


Submitted by: Chan Chung [[log in to unmask]]
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The STE group kinase SepA controls cleavage furrow formation in  
Dictyostelium

Annette Müller-Taubenberger*, Hellen C. Ishikawa-Ankerhold, Peter M.  
Kastner,
Emmanuel Burghardt, and Günther Gerisch


Cell Motility and the Cytoskeleton, in press.

During a REMI screen for proteins regulating cytokinesis in  
Dictyostelium
discoideum we isolated a mutant forming multinucleate cells. The gene
affected in this mutant encoded a kinase, SepA, which is an ortholog of
Cdc7, a serine-threonine kinase essential for septum formation in
Schizosaccharomyces pombe. Localization of SepA-GFP in live cells and
its presence in isolated centrosomes indicated that SepA, like its  
upstream
regulator Spg1, is associated with centrosomes. Knockout mutants of SepA
showed a severe cytokinesis defect and a delay in development. In
multinucleate SepA-null cells nuclear division proceeded normally and
synchronously. However, often cleavage furrows were either missing or
atypical: they were extremely asymmetric and constriction was impaired.
Cortexillin-I, a marker localizing strictly to the furrow in wild-type  
cells,
demonstrated that large, crescent-shaped furrows expanded and persisted
long after the spindle regressed and nuclei returned to the interphase  
state.
Outside the furrow the filamentous actin system of the cell cortex  
showed
strong ruffling activity. These data suggest that SepA is involved in  
the
spatial and temporal control system organizing cortical activities in
mitotic and post-mitotic cells.


Submitted by: Annette Müller-Taubenberger [[log in to unmask]]
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[End dictyNews, volume 32, number 12]