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doi: 10.1242/10.1242/dev.00248

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Mbt, a Drosophila PAK protein, combines with Cdc42 to regulate photoreceptor cell morphogenesis

University of Würzburg, Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Versbacherstr. 5, 97078 Würzburg, Germany

View larger version (194K): [in a new window]   Fig. 1. Adult mbt eye phenotype. Tangential sections through wild-type (A) and mbtP1 (B) eyes. In the wild type, each ommatidium contains eight photoreceptor cells. R1-R6 have large rhabdomeres, the central R7 and R8 (not seen in apical sections) rhabdomeres are small. Longitudinal sections through wild-type (C) and mbtP1 (D) eyes show the defects in proximodistal rhabdomere extension in the mutant. Scale bar: 10 µm.

View larger version (94K): [in a new window]   Fig. 2. The mbt phenotype in third instar eye imaginal discs. Shown are apical-to-basal projection views of wild-type (A,C,E) and mbtP1 (B,D,F) eye imaginal discs stained with anti-Mbt (A,B) or anti-Arm (C,D). A merge of both stains is seen in E,F. Below each projection view, an apical-to-basal cross-section is shown (the bracket indicates the apical-basal extension of the eye disc). Photoreceptor cell differentiation starts in the morphogenetic furrow and involves the step-wise recruitment of eight photoreceptor cells in a single ommatidium. In the wild type, Mbt and Arm colocalise at the AJs of all photoreceptor cells as soon as they become integrated into the ommatidial clusters (E). Despite the absence of detectable levels of Mbt protein in mbtP1 mutant eye discs (B), AJs between photoreceptor cells do form in the mutant, although some irregularities are already evident (arrow in D). (G,H) Anti-Elav staining of wild-type (G) and mbtP1 mutant eye discs (H). Occasionally, mbtP1 ommatidia contain fewer differentiating photoreceptor cells (arrow). The morphogenetic furrow is on the left-hand side in all figures. Scale bar: 10 µm.

View larger version (98K): [in a new window]   Fig. 3. Mbt mutations disrupt adherens junction integrity during pupal photoreceptor cell morphogenesis. Shown are proximal-to-distal projection views of wild-type (WT) eye imaginal discs at 37% p.d. (left column) and 50% p.d. (middle column) stained with anti-Mbt and anti-Arm antibodies. Staining of both overlap in the developing photoreceptor cells and also in the surrounding pigment and bristle cells (WT merge). Three-dimensional reconstructions of single ommatidial clusters at 37% p.d. (left column, insets) and 50% p.d. (right column) illustrate the massive distal (top)-to-proximal (bottom) extension of the AJs during pupal development. Anti-Arm staining of mbtP1 pupal eye discs (mbtP1, -Arm) shows the disorganisation of the AJs. In the proximal-to-distal projection view, lateral extensions are evident (arrow) and the AJs fail to extend (inset). At 50% p.d., Armadillo accumulates in patches (right picture). Scale bars: 10 µm.

View larger version (82K): [in a new window]   Fig. 4. Staining of wild-type and mbtP1 ommatidia (50% p.d.) with anti-Crb (A,B) and anti-Dlg (C,D) antibodies. Below each proximal-to-distal projection view, the corresponding proximal-to-distal cross-section is shown. Scale bar: 5 µm.

View larger version (46K): [in a new window]   Fig. 5. (A) Binding of Mbt to Rho-type GTPases. HA-tagged Mbt or MbtH19,22L were transiently expressed in HEK293 cells alone or co-expressed with Myc-tagged Cdc42, Rac1 or Rho1, or the corresponding activated forms. Cell lysates (Lysates) were analysed using western blot (WB) with anti-HA or anti-Myc antibodies to indicate expression of the corresponding proteins. For co-immunoprecipitation, cell lysates were incubated with an anti-HA antibody and GTPases bound to immunopurified Mbt were detected by western blot using the anti-Myc antibody (IP, -HA; WB, -Myc). The presence of Mbt in the immunoprecipitates was verified by western blot (IP, -HA; WB, -HA). The binding of Cdc42 and Cdc42G12V to Mbt is disrupted by mutation of the PBD (MbtH19,22L). (B) Mbt interacts with Cdc42 in a GTP-dependent manner. The protein extract from HEK293 cells transiently transfected with the pcDNA3-HA-Mbt construct was incubated either with unloaded, or with GDP- or GTP-loaded GST-Cdc42 protein. The GST protein and any bound cellular proteins were recovered by precipitation with glutathione-sepharose beads and separated by SDS-PAGE. Mbt protein was detected by western blot using the anti-HA antibody. As a control, total lysates from pcDNA3-HA-Mbt transfected HEK293 cells are shown (input). The presence of equal amounts of the GST-Cdc42 was verified with an anti-GST antibody. (C) Sequence alignment of the p21-binding domain (PBD) and the kinase inhibitory domain (KID) of human PAK1 and D-PAK (group I PAKs) with the corresponding sequences of human PAK4, PAK5, PAK6 and Mbt (group II PAKs). Highly conserved amino acids are indicated in light grey, amino acids only conserved between PAK1/D-PAK or PAK4-6/Mbt are shown in black and dark grey, respectively. The serine 144 autophosphorylation site and lysine 141 (black circles), which are involved in regulating PAK1 kinase activity are not present in PAK4-6/Mbt. The histidine residues that were mutated to create a Cdc42 binding-deficient Mbt protein are indicated by triangles. (D) Regulation of Mbt kinase activity. HEK293 cells were transfected with either an empty pcDNA3 vector (control), with HA-tagged Mbt, MbtH19,22L or MbtT525A alone or in combination with Myc-tagged Cdc42 or Cdc42G12V. Expression of Mbt was verified by western blot (WB). Mbt proteins were immunopurified from cell lysates using an anti-HA antibody and incubated in kinase buffer with myelin basic protein (MBP) as a substrate in the presence of [-32P]ATP. Autophosphorylation of Mbt and MBP phosphorylation were analysed by autoradiography.

View larger version (65K): [in a new window]   Fig. 6. Rescue of the mbt eye phenotype by transgenic constructs. Tangential sections of eyes of the following genotypes: (A) mbtP1/Y; UAS:mbt/+; Gal4:238Y/+; (B) mbtP1/Y; UAS:mbtT525A/+; Gal4:238Y/+; and (C) mbtP1/Y; UAS:mbtH19,22L/+; Gal4:238Y/+. Expression of the mbt cDNA with Gal4 line 238Y in a mbtP1 mutant background results in a complete rescue of the mbtP1 eye phenotype (A, compare with Fig. 1B). MbtT525A can only partially substitute the loss of endogenous Mbt function (B); occasionally photoreceptor cells are missing (arrows). No rescue of the mbtP1 phenotype is observed upon expression of the mbtH19,22L construct (C). Scale bar: 10 µm.

View larger version (87K): [in a new window]   Fig. 7. The Cdc42-binding domain is required for AJ localisation of the Mbt protein. Pupal eye discs (50% p.d.) from mbtP1 animals expressing transgenic Mbt (A-C), MbtT525A (D-F), or MbtH19,22L (G-I) with Gal4:238Y were co-stained with the anti-Mbt (A,D,G) and anti-Arm (B,E,H). A merge of the staining for both is seen in C, F and I. Below each proximal to distal projection view, a proximal-to-distal cross-section is shown. The transgenic Mbt and MbtT525A proteins colocalise with Arm at AJs of the photoreceptor cells (A-C and D-F, respectively). In contrast to the non-mutated Mbt protein, the MbtT525A protein is unable to rescue the mbtP1 AJ defects fully (compare B with E). Mutation of the Cdc42-binding site leads to cytoplasmic distribution of the MbtH19,22L protein (G). As revealed by anti-Arm staining, the MbtH19,22L protein cannot rescue the mbtP1 AJs defects fully (H). (K-M) Expression of MbtH19,22L in a wild-type background. Note that the Mbt antibody recognises both the endogenous and the MbtH19,22L protein in this preparation. Scale bar: 10 µm.

View larger version (88K): [in a new window]   Fig. 8. Influence of Cdc42 on localisation of the Mbt protein. (A-I) Two ommatidial clusters of an eye imaginal disc derived from an animal of the genotype Cdc424, FRT 19A/tubP-Gal80, hs-Flp, FRT19A; GMR-Gal4/UAS: mCD8-GFP are shown. The eye disc was stained with the anti-Mbt serum (red) and the anti-mCD8 antibody (green). Heat-shock induced Flippase (Flp) activity produces recombination between the FRT19A bearing chromosomes resulting in daughter cells, which are homozygous for the cdc424 mutation and at the same time lack the Gal4 suppressor Gal80. This allows expression of the GMR-Gal4-driven mCD8-GFP reporter gene in homozygous cdc424 cells. Apical-to-basal projection views of an eye disc containing a single cdc424 mutant photoreceptor cell are shown in A (anti-Mbt), B (anti-mCD8) and C (merge); D-F focus on the most apical domain; G-I are apicobasal cross-sections. The cdc424 mutant cell is devoid of detectable levels of Mbt protein (arrowheads), while the other photoreceptor cells express high levels of apical localised Mbt protein. The arrow indicates to the axonal projection. (K-N) Localisation of Mbt and Arm in third instar eye imaginal discs upon expression of Cdc42T17N (K,L) and Cdc42G12V (M,N). Scale bars: 5 µm in I; 10 µm in N.