dictyNews
Electronic Edition
Volume 39, number 32
November 2, 2013

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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), 
Jörn Linkner (1), Michael Schleicher (2), and Jan Faix (1)

(1) Institute for Biophysical Chemistry, Hannover Medical School, 
Carl-Neuberg Straße 1, 30625 Hannover, Germany; 
(2) Institute for Anatomy and Cell Biology, Ludwig-Maximilians-University, 
80336 München, 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 [[log in to unmask]]
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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 [[log in to unmask]]
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[End dictyNews, volume 39, number 32]