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First published online 2 February 2005
doi: 10.1242/dev.01669

Development 132, 1009-1020 (2005)
Published by The Company of Biologists 2005
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RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation

Jörg Großhans1,*,{dagger}, Christian Wenzl1,*, Hans-Martin Herz1, Slawomir Bartoszewski1, Frank Schnorrer2, Nina Vogt2, Heinz Schwarz2 and H.-Arno Müller3

1 ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany
2 Max-Planck-Institut für Entwicklungsbiologie, Spemannstraße 35, 72076 Tübingen, Germany
3 Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1 Geb. 26.02., 40225 Düsseldorf, Germany



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  		Fig. 1. Dilated furrow canals in embryos from RhoGEF2 and dia germline clones. (A) Schematic drawing of cellularisation showing the furrow canal, basal junction and plasma membrane. Embryos prepared for TEM by high pressure freezing and freeze substitution. (B) In the wild-type embryo, furrow canals are seen as a dilatation of the plasma membrane growing in from the apical surface. In embryos from (C,D) RhoGEF21.1 and (E,F) dia germline clones, furrow canals are considerably enlarged and filled with large cytoplasmic blebs (arrows in C,D,E). No simple hairpin structure can be seen in most of the cases. Apical side up. Scale bars: 0.5 µm.

 


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  		Fig. 2. Multinuclear cells in embryos from RhoGEF2 and dia germline clones. (A-E) Embryos of the following genetic constitution were stained for DNA (blue), {gamma}-tubulin (green, added from a higher optical section) and F-actin (red). (A) Wild type, (B) from RhoGEF21.1 germline clones, (C) nullo, (D) from dia germline clones and (E) slam embryos from slam germline clones. Number of embryos with indicated proportion of nuclei in multinuclear cells plotted for (F) RhoGEF2 alleles (1.1, 4.1, 04291) and for (G) dia. Scale bars: 10 µm.

 


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  		Fig. 3. RhoGEF2 and nullo/sry-{alpha} cooperate in furrow canal formation. (A-E) Embryos from RhoGEF21.1 germline clones were injected at the posterior pole with the following dsRNA: (A) Bsg25D, (B,E) nullo, (C,D) sry-{alpha}, and stained for DNA (blue), F-actin (green), Sry-{alpha} (red). Loss of Sry-{alpha} protein and loss of furrow canals (marked by F-actin, optical transversal sections, D,E) correlates with the posterior site of dsRNA injection as shown by respective antibody staining (C). Scale bars: (A-C) 50 µm, (D,E) 10 µm. Posterior to the right, arrow indicates injection site.

 


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  		Fig. 4. RhoGEF2 is enriched at the furrow canal. Embryos (heatfixed) stained with RhoGEF2 antibody (green, white), Dlg (red) and DNA (blue). Right half of A-D shows only the RhoGEF2 staining. (A-C,E,F) Wild type, (D) from RhoGEF204291 germline clone. (E) Surface view; wild type. (F) Cross section of an embryo undergoing ventral furrow formation. Scale bar: (A-E) 10 µm, (F) 20 µm. (G) Western blot for RhoGEF2 or Dlg of extracts from wild-type embryos and embryos from RhoGEF204291 germline clones.

 


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  		Fig. 5. RhoGEF2 colocalises with Dia and F-actin, but not Arm. Double labelling of wild-type embryos in green for (A,C,D) RhoGEF2, (B,E) Dia and in red for (A,B) Arm, (C) Dia and (E,F) F-actin. (A-C) heat-fixed, (D,E) fixed with formaldehyde. Scale bar: 10 µm.

 


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  		Fig. 6. RhoGEF2 and Dia localisation at the furrow canal do not depend on microfilaments. Permeabilized wild-type (A,B) embryos or (C) embryos with a GFP-moesin transgene were incubated with (A) buffer or (B,C) latrunculin A (LatA) to inhibit actin polymerisation, and stained for DNA (blue), F-actin (green); (A,B) RhoGEF2 and Dia; (C) GFP fluorescence. Scale bar: 50 µm.

 


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  		Fig. 7. F-actin and Dia localisation depend on RhoGEF2. (A,C,E) Wild-type embryos and embryos from (B,F) RhoGEF21.1 and (D) dia germline clones stained for (A-D) F-actin (left, white), (E,F) Dia (left, white), Sry-{alpha} (red) and DNA (blue). Sry-{alpha} staining indicates equal staining conditions. (G) Western blot for Dia and {alpha}-tubulin with extracts from wild-type, RhoGEF21.1 or dia germline clones embryos. Scale bar: 10 µm.

 


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  		Fig. 8. Rho1 is a substrate of RhoGEF2 and binds and activates Dia. (A) Release of tritium-loaded GDP from Rho1 and Rac1 GST fusion proteins by a fusion protein of GST and the GEF domain from RhoGEF2. Bound GDP (expressed as counts of radioactivity per minute; cpm) plotted against incubation time. (B) Specificity of GDP release. Tritium loaded GST fusion proteins were incubated with GST fusion proteins of the GEF domains of RhoGEF2 and Trio (domain 1) or GST for 20 minutes. Bound GDP is indicated as the quotient of [GDP](t=0 minutes) and [GDP](t=20 minutes). The protein T1544A has a single point mutation in the GEF domain of RhoGEF2. (C) GST, GST-Rho1, GST-Rho1Q63L, GST-Rac1, (loaded with GDP or GMP-PNP) bound to glutathione Sepharose were incubated with reticulocyte lysate containing 35S-labelled Dia{Delta}N318 or Dia{Delta}C464. Rho1Q63L has no GTPase activity. Bound and unbound fractions were analysed by SDS-PAGE and autoradiography. (D) Actin polymerisation induced by Dia. Time course of fluorescence (relative units) of pyrene-labelled actin (3 µM) and the indicated components. Dia FH, ZZ-Dia{Delta}N519, Dia {Delta}FH, ZZ-Dia{Delta}C518, Rho1, GST-Rho1 loaded with GTP{gamma}S. Rho1 loaded with GDP or GMP-PNP or Rho1Q63L released autoinhibition to a similar degree.

 

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© The Company of Biologists Ltd 2005