dictyNews
Electronic Edition
Volume 36, number 8
March 11, 2011
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Abstracts
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An Extracellular Matrix, Calmodulin-Binding Protein from Dictyostelium
with EGF-Like Repeats that Enhance Cell Motility
Andres Suareza, Robert Huberb, Michael Myrec, Danton H. O’Daya,b,*
aDepartment of Biology, University of Toronto at Mississauga, Mississauga,
Ontario, Canada
bDepartment of Cell and Systems Biology, University of Toronto, Toronto,
Ontario, Canada
cMolecular Neurogenetics Unit, Center for Human Genetic Research,
Massachusetts General Hospital, Boston, Massachusetts, USA
Cellular Signalling, in press
CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated
using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting
it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into
two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding
domain was detected and both CaM-agarose binding and CaM
immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in
both a Ca2+-dependent and -independent manner. cyrA-C45 was present
continuously throughout growth and development but was secreted at high
levels during the multicellular slug stage of Dictyostelium development. At this
time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified
the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first
EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and
-C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here
we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related
to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis.
The status of cyrA as an extracellular CaMBP was further clarified by the
demonstration that CaM is secreted during development. Antagonism of CaM
with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for
extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first
extracellular CaMBP identified in Dictyostelium and since it is an ECM protein
with EGF-like repeats that enhance cell motility it likely also represents the first
matricellular protein identified in a lower eukaryote.
Submitted by Danton O’Day [[log in to unmask]]
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Functional analysis of Dictyostelium IBARa suggests a conserved role of
the I-BAR domain in endocytosis
Douwe M. Veltman, Giulio Auciello, Heather J. Spence, Laura M. Machesky,
Joshua Z. Rappoport and Robert H. Insall
Biochemical Journal, in press
I-BAR domain containing proteins such as IRSp53 associate with outwardly
curved membranes and connect them to proteins involved in actin dynamics.
Research on I-BAR proteins has focussed on possible roles in filopod and
lamellipod formation, but their full physiological function remains unclear.
The social amoeba Dictyostelium encodes a single I- BAR/SH3 protein,
called IBARa, along with homologues of proteins that interact with IRSp53
family proteins in mammalian cells, providing an excellent model to study
its cellular function. Disruption of the gene encoding IBARa leads to a mild
defect in development, but filopod and pseudopod dynamics are unaffected.
Furthermore, ectopically expressed IBARa does not induce filopod formation
and does not localize to filopods. Instead, IBARa associates with clathrin
puncta immediately before they are endocytosed. This role is conserved-
human BAIAP2L2 also tightly colocalizes with clathrin plaques, though its
homologues IRSp53 and IRTKS associate with other punctate structures.
Our data suggest that I-BAR-containing proteins help generate the membrane
curvature required for endocytosis and implies an unexpected role for IRSp53
family proteins in vesicle trafficking.
Submitted by Robert Insall [[log in to unmask]]
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Actin Polymerization Driven by WASH Causes V-ATPase Retrieval and
Vesicle Neutralization Before Exocytosis.
Michael Carnell, Tobias Zech, Simon Johnston, Robin May, Thierry Soldati,
Laura Machesky and Robert Insall
J. Cell Biol., in press
WASH is a recently identified and evolutionarily conserved regulator of actin
polymerization. Here we show that WASH coats mature Dictyostelium
lysosomes and is essential for exocytosis of indigestible material. A related
process, expulsion of the lethal endosomal pathogen Cryptococcus
neoformans from mammalian macrophages, also uses WASH-coated
vesicles and cells expressing dominant negative WASH mutants inefficiently
expel Cryptococcus. Dictyostelium WASH causes F-actin patches to form
on lysosomes, leading to removal of vacuolar ATPase (V-ATPase) and
neutralization of lysosomes to form postlysosomes. Without WASH, no
patches or coat are formed, neutral postlysosomes are not seen, and
indigestible material such as dextran is not exocytosed. Similar results
occur when actin polymerization is blocked with latrunculin. V-ATPases
are known to bind avidly to F-actin. Our data imply a new mechanism,
actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize
actin on vesicles to drive separation and recycling of proteins such as the
V-ATPase.
Submitted by Robert Insall [[log in to unmask]]
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Modelling cell movement and chemotaxis using pseudopod-based
feedback.
M.P. Neilson, J.A. Mackenzie, S.D. Webb and R.H. Insall
SIAM journal on Scientific Computation, in press
A computational framework is presented for the simulation of eukaryotic
cell migration and chemotaxis. An empirical pattern formation model,
based on a system of non-linear reaction-diffusion equations, is
approximated on an evolving cell boundary using an Ar- bitrary
Lagrangian Eulerian surface finite element method (ALE-SFEM). The
solution state is used to drive a mechanical model of the protrusive and
retractive forces exerted on the cell boundary. Movement of the cell is
achieved using a level set method. Results are presented for cell
migration with and without chemotaxis. The simulated behaviour is
compared with experimental results of migrating Dictyostelium
discoideum cells.
Submitted by Robert Insall [[log in to unmask]]
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A Polarized Epithelium Organized by Beta- and Alpha-Catenin Predates
Cadherin and Metazoan Origins
Daniel J. Dickinson, W. James Nelson,* William I. Weis*
*To whom correspondence should be addressed
Science, 11 March 2011, Vol. 331, #6022
A fundamental characteristic of metazoans is the formation of a simple,
polarized epithelium. In higher animals, the structural integrity and
functional polarization of simple epithelia require a cell-cell adhesion
complex containing a classical cadherin, the Wnt-signaling protein
beta-catenin and the actin-binding protein alpha-catenin. We show that
the non-metazoan Dictyostelium discoideum forms a polarized
epithelium that is essential for multicellular development. Although
D. discoideum lacks a cadherin homolog, we identify an alpha-catenin
ortholog that binds a beta-catenin-related protein. Both proteins are
essential for formation of the epithelium, polarized protein secretion
and proper multicellular morphogenesis. Thus the organizational
principles of metazoan multicellularity may be more ancient than
previously recognized, and the role of the catenins in cell polarity
predates the evolution of Wnt signaling and classical cadherins.
Submitted by Daniel Dickinson [[log in to unmask]]
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[End dictyNews, volume 36, number 8]
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