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dictyNews Volume 39 Number 32
dictyNews
Electronic Edition
Volume 39, number 32
November 2, 2013
Please submit abstracts of your papers as soon as they have been
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Abstracts
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The Diaphanous-related formin dDia1 is required for highly directional
phototaxis and formation of properly sized fruiting bodies in Dictyostelium
Moritz Winterhoff (1), Alexander Junemann (1), Benjamin Nordholz (1),
Jrn Linkner (1), Michael Schleicher (2), and Jan Faix (1)
(1) Institute for Biophysical Chemistry, Hannover Medical School,
Carl-Neuberg Stra§e 1, 30625 Hannover, Germany;
(2) Institute for Anatomy and Cell Biology, Ludwig-Maximilians-University,
80336 Mnchen, Germany
EJCB, in press
Diaphanous-related formins (DRFs) act as downstream effectors of Rho
family GTPases and drive the formation and elongation of linear actin
filaments in various cellular processes. Here we analyzed the DRF dDia1
from Dictyostelium cells. The biochemical characterization of recombinant
dDia1-FH1FH2 by bulk polymerization assays and single filament TIRF
microscopy revealed that dDia1 is a rather weak nucleator. Addition of
any of the three Dictyostelium profilin isoforms, however, markedly
accelerated formin-mediated actin filament barbed end elongation in TIRF
assays. Interestingly, filament elongation was significantly faster in presence
of DdPFN I (profilin I) when compared to the other two isoforms, suggesting
selectivity of dDia1 for DdPFN I. Additionally, we frequently observed
dissociation of the formin from growing barbed ends. These findings are
consistent with dilution-induced depolymerization assays in presence of
dDia1-FH1FH2 showing that dDia1 is a weak capper in comparison with
heterodimeric capping protein. To study the physiological role of this formin,
we created cell lines lacking dDia1 or overexpressing GFP-tagged dDia1.
Of note, constitutively active dDia1 accumulated homogenously in the entire
pseudopod suggesting that it controls microfilament architecture to regulate
cell migration. Comparison of wild type and dDia1-null cells in random cell
migration and chemotaxis towards a cAMP gradient revealed no major
differences. By contrast, phototaxis of dDia1-deficient cells during the
multicellular stage was markedly impaired. While wild type slugs moved
with high directionality towards the light source, the trails of dDia1-null slugs
displayed a characteristic V-shaped profile and deviated in angles between
50¡-60¡ from the path of the incident light. Possibly in conjunction with this
defect, dDia1-null cells also formed substantially smaller fruiting bodies.
These findings demonstrate dDia1 to be critically involved in collective cell
migration during terminal differentiation.
Submitted by Jan Faix [faix.Jan@mh-hannover.de]
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A Dictyostelium cellobiohydrolase orthologue that affects
developmental timing
Mizuho Kunii 1, Mami Yasuno, Yuki Shindo and Takefumi Kawata *
Department of Biology, Faculty of Science, Toho University, 2-2-1
Miyama, Funabashi, Chiba 274-8510, Japan
1 Present address: Department of Biological Production, Graduate
School of Agriculture, Tokyo University of Agriculture and Technology,
3-8-1 Harumi-cho, Fuchu, Tokyo 183-8538, Japan
Development Genes and Evolution, in press
Dictyostelium discoideum is a facultative multicellular amoebozoan with
cellulose in the stalk and spore coat of its fruiting body as well as in the
extracellular matrix of the migrating slug. The organism also harbors a
number of cellulase genes. One of them, cbhA, was identified as a
candidate cellobiohydrolase gene based on the strong homology of its
predicted protein product to fungal cellobiohydrolase I (CBHI). Expression
of the cbhA was developmentally regulated, with strong expression in the
spores of the mature fruiting body. However, a weak but detectable level
of expression was observed in the extracellular matrix at the mound -
tipped finger stages, in prestalk O cells, and in the slime sheath of the
migrating slug - late culminant stages. A null mutant of the cbhA showed
almost normal morphology. However, the developmental timing of the
mutant was delayed by 2 - 4 hours. When a c-Myc epitope-tagged CbhA
was expressed, it was secreted into the culture medium and was able to
bind crystalline cellulose. The CbhA-myc protein was glycosylated, as
demonstrated by its ability to bind succinyl concanavalin A-agarose.
Moreover, conditioned medium from the cbhA-mycoe strain displayed
4-methylumbelliferyl beta-D-cellobioside (4-MUC) digesting activity in
Zymograms in which conditioned medium was examined via native-
polyacrylamide gel electrophoresis or spotted on an agar plate containing
4-MUC, one of the substrates of cellobiohydrolase. Taken together, these
findings indicate that Dictyostelium CbhA is an orthologue of CBH I that
is required for a normal rate of development.
Submitted by Tekefumi Kawata [tkawata@bio.sci.toho-u.ac.jp]
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[End dictyNews, volume 39, number 32]
