Copy Link
Add to Bookmark
Report
dictyNews Volume 35 Number 01
dictyNews
Electronic Edition
Volume 35, number 1
July 2, 2010
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.
Follow dictyBase on twitter:
http://twitter.com/dictybase
=========
Abstracts
=========
A versatile set of tagged expression vectors to monitor protein localisation
and function in Dictyostelium.
Manu Dubin, Wolfgang Nellen,
Abt. Genetik, Universität Kassel, 34132 Kassel, Germany
Gene, in press
We describe here a series of vectors for ectopic expression of tagged
proteins in Dictyostelium discoideum. These vectors allow the addition
of N- or C- terminal tags (GFP, mRFP, 3xFLAG, 3xHA, 6xMYC or TAP)
with an optimised polylinker sequence and no additional amino acid
residues at the N- or C-terminus of the protein. The expression cassettes
were introduced into vectors containing Blasticidin or Geneticin resistance
markers and into integrating as well as extrachromosomal plasmids.
The vectors are designed as high and low copy versions and thus allow
for a limited expression level control. They are also convenient with
regard to complementation, co- and super-transformation. Finally the
vectors share standardizsed cloning sites, so that a gene of interest
can be easily transferred between vectors as experimental requirements
evolve. These vectors were used to study the localisation of several
putative RNA processing proteins including EriA and DicerB.
Submitted by Wolfgang Nellen [nellen@uni-kassel.de]
--------------------------------------------------------------------------------
Synergy between two transcription factors directs gene expression in
Dictyostelium tip-organiser cells
Hong Yu Wang and Jeffrey G. Williams
College of Life Sciences, University of Dundee , Dow St.,
Dundee DD1 5EH, U. K.
Int J Dev Biol, in press
cotC requires the transcription factor CudA for its expression in the
posterior, prespore cells of the slug while the expL7 gene requires
CudA for its expression in the anterior, tip-organiser region. In order to
identify additional transcription factors that might mediate tip-organiser
specific expression, we performed affinity chromatography on slug
nuclear extracts. The affinity matrix bore cap-site distal sequences
from region A’ of the expL7 promoter; an essential region located
upstream of the CudA binding domain. One of the proteins purified
was GBF, a zinc finger transcription factor which binds to G-rich
elements, known as G boxes, that are present in the promoters of
many developmental genes, including cotC. Previous work identified
an essential sequence motif within region A’ and we show that this
element is a G box, that binds recombinant GBF. Moreover, a G box
from within the cotC promoter can substitute for region A’ of expL7 in
directing tip-organiser specific expression of expL7. Thus the same
two transcription factors, CudA and GBF, seem to co-operate to direct
both tip-organiser and prespore gene expression. How then is
specificity achieved? Replacing a CudA binding region in the cotC
promoter with the CudA binding domain from expL7 strongly represses
cotC promoter activity. Hence we suggest that differences in the
topology of the multiple CudA half- sites contained within the two
different CudA binding regions, coupled with differences in the
signalling environment between tip-organiser cells and prespore cells,
ensure correct expL7 expression.
Submitted by Jeff Williams [j.g.williams@dundee.ac.uk]
--------------------------------------------------------------------------------
Analysis of Dictyostelium TACC reveals differential interactions with
CP224 and unusual dynamics of Dictyostelium microtubules
Matthias Samereier, Otto Baumann, Irene Meyer and Ralph Gräf*
University of Potsdam, Institute for Biochemistry and Biology,
Dept. of Cell Biology, Karl-Liebknecht-Strasse 24-25, Haus 26,
14476 Potsdam-Golm, Germany
Cellular and Molecular Life Science, in press
We have localized TACC to the microtubule-nucleating centrosomal
corona and to microtubule plus ends. Using RNAi we proved that
Dictyostelium TACC promotes microtubule growth during interphase
and mitosis. For the first time we show in vivo that both TACC and
XMAP215 family proteins can be differentially localized to microtubule
plus ends during interphase and mitosis and that TACC is mainly
required for recruitment of an XMAP215-family protein to interphase
microtubule plus ends but not for recruitment to centrosomes and
kinetochores. Moreover, we have now a marker to study dymamics
and behavior of microtubule plus ends in living Dictyostelium cells.
In a combination of live cell imaging of microtubule plus ends and
fluorescence recovery after photobleaching (FRAP) experiments of
GFP-alpha-tubulin cells we show that Dictyostelium microtubules
are dynamic only in the cell periphery, while they remain stable at
the centrosome, which also appears to harbor a dynamic pool of
tubulin dimers.
Submitted by Ralph Gräf [rgraef@uni-potsdam.de]
------------------------------------------------------------------------------
The exocytic gene secA is required for Dictyostelium cell motility
and osmoregulation
Roberto Zanchi, Gillian Howard, Mark S. Bretscher and Robert R. Kay
MRC Laboratory of Molecular Biology, Hills Road, Cambridge,
CB2 0QH, UK
Journal of Cell Science, in press
We investigated the link between plasma membrane recycling and
cell movement using a fast-acting, temperature sensitive mutant of
the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells
become almost paralyzed within minutes at the restrictive temperature.
However, they can still sense cyclic-AMP gradients and polymerize
actin up-gradient, but form only abortive pseudopodia, which cannot
expand. They also relay a cyclic-AMP signal normally, suggesting
that cyclic-AMP is released by a non-exocytic mechanism. To
investigate why SecA is required for motility, we examined membrane
trafficking in the mutant. Plasma membrane circulation is rapidly
inhibited at the restrictive temperature and the cells acquire a
prominent vesicle. Organelle-specific markers show this is an
un-discharged contractile vacuole and we find the cells are
correspondingly osmo-sensitive. Electron microscopy shows that
many smaller vesicles, probably originating from the plasma
membrane, also accumulate at the restrictive temperature.
Consistent with this, the surface area of mutant cells shrinks. We
suggest that SecA mutant cells cannot move at the restrictive
temperature because their block in exocytosis results in a net
uptake of plasma membrane, reducing its area, and so restricting
pseudopodial expansion. This demonstrates the importance of
proper surface area regulation in cell movement.
Submitted by Rob Kay [rrk@mrc-lmb.cam.ac.uk]
------------------------------------------------------------------------------
The following article has been included because it contains experiments in
human cells and Dictyostelium
Strumpellin is a Novel VCP Binding Protein linking Hereditary Spastic
Paraplegia to Protein Aggregation Diseases.
Christoph S. Clemen1),#, Karthikeyan Tangavelou1),#, Karl-Heinz
Strucksberg1), Steffen Just2), Linda Gaertner2), Hanna Regus-Leidig3),
Maria Stumpf1), Jens Reimann4), Roland Coras5), Reginald O. Morgan6),
Maria-Pilar Fernandez6), Andreas Hofmann7), Stefan Müller8), Benedikt
Schoser9), Franz-Georg Hanisch8), Wolfgang Rottbauer2,10), Ingmar
Blümcke5), Stephan von Hörsten11), Ludwig Eichinger1), Rolf Schröder5)
1) Institute of Biochemistry I, Medical Faculty, University of Cologne,
Cologne, Germany
2) Department of Medicine III, University of Heidelberg, Heidelberg, Germany
3) Department of Biology, Animal Physiology, University of
Erlangen-Nuremberg, Erlangen, Germany
4) Department of Neurology, University Hospital Bonn, Bonn, Germany
5) Institute of Neuropathology, University Hospital Erlangen, Erlangen,
Germany
6) Department of Biochemistry and Molecular Biology, University of Oviedo
and University Institute of Biotechnology of Asturias, Oviedo, Spain
7) Structural Chemistry Program, Eskitis Institute for Cell & Molecular
Therapies, Griffith University, Brisbane, Australia
8) Institute of Biochemistry II, Medical Faculty, and Center for Molecular
Medicine Cologne, University of Cologne, Cologne, Germany
9) Friedrich-Baur Institute, Department of Neurology, Ludwig-Maximilians
University of Munich, Munich, Germany
10) Department of Medicine II, University of Ulm, Ulm, Germany
11) Franz-Penzoldt-Center, Experimental Therapy,
Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
# Both authors contributed equally to this work.
Brain, in press
Mutations of the human VCP gene cause autosomal-dominant inclusion
body myopathy associated with Paget disease of bone and frontotemporal
dementia (IBMPFD). We identified strumpellin as a novel VCP binding
partner. Strumpellin mutations have been shown to cause hereditary spastic
paraplegia. We demonstrate that strumpellin is a ubiquitously expressed
protein present in cytosolic and endoplasmic reticulum cell fractions.
Over-expression or ablation of wild-type strumpellin caused significantly
reduced wound closure velocities in wound healing assays, whereas
over-expression of the disease causing strumpellin N471D mutant showed
no functional effect. Strumpellin knock-down experiments in human
neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth.
Knock-down studies in zebrafish revealed severe cardiac contractile
dysfunction, tail curvature and impaired motility. The latter phenotype is
due to a loss of central and peripheral motoneuron formation. These data
imply a strumpellin loss-of-function pathogenesis in hereditary spastic
paraplegia. In the human central nervous system strumpellin shows a
pre-synaptic localization. We further identified strumpellin in pathological
protein aggregates in IBMPFD, various myofibrillar myopathies,
and in cortical neurons of a Huntington disease mouse model. Beyond
hereditary spastic paraplegia, our findings imply that mutant forms of
strumpellin and VCP may have a concerted pathogenic role in various
protein aggregate diseases.
Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de]
==============================================================
[End dictyNews, volume 35, number 1]
