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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00663

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Kette regulates actin dynamics and genetically interacts with Wave and Wasp

Institut für Neurobiologie, Universität Münster, Badestr. 9, D-48149 Münster, Germany

View larger version (50K): [in a new window]   Fig. 1. Kette associates with membranes. (A) Schematic view of the Kette protein; hydrophobic domains are indicated. Different antisera were generated. N, N-terminal domain, amino acids 1-374; the M-serum directed amino acids 375-907 is not indicated; C, C-terminal domain, amino acids 908-1126; P, peptide antibodies, amino acids 652-666. (B) Western blot analyses of protein extracts prepared from wild-type, rhoGAL4/UAS-Kette full-length or rhoGAL4/UAS-Kettemyc embryos were probed either with the anti-Kette P antiserum or anti-Myc antibodies (Mab 9E10) as indicated. The Kette protein is 120 kDa. Anti-Myc antibodies recognize a similar sized protein confirming the specificity of the antisera. (C) Differential centrifugation reveals that Kette is located primarily in the cytosol. Western blots were probed with anti-Kette antisera (top), anti-actin antibodies (middle) and anti Na+/K+ ATPase antibodies (bottom) to monitor a typical transmembrane protein. The different lanes show: total, total protein extracts of S2 cells; PNS, supernatant following 1,000 g centrifugation. The supernatant was subjected to 25,000 g centrifugation and subsequently to 200,000 g centrifugation. The pellet was resuspended in either PBS at high pH or 1% NP-40 and subjected to 200,000 g centrifugation (S, supernatant; P, pellet). For each lane, equal amounts of total proteins were loaded on SDS-PAGE. (D) Post-nuclear supernatant (PNS) of wild-type embryos was subjected to equilibrium sedimentation on a discontinuous sucrose density gradient. Only small amounts of the Kette protein were detectable in the membrane fraction, while most of the Kette protein remains in high dense sucrose containing cytosolic proteins. Western blot analysis was performed using anti-Kette antisera (top) and anti Fas3 antibodies (bottom) to monitor a typical transmembrane protein. Equal amounts of total proteins in the post nuclear supernatant (PNS), membrane and cytosolic fraction were loaded for SDS-PAGE.

View larger version (44K): [in a new window]   Fig. 4. Kette expression. (A) Top panel shows a western Blot probed for Kette expression (P-antiserum), equal amounts of protein were loaded on the gel (Coomassie stain). (B) Western blot showing Kette expression in S2 cells and similarly increased levels of Kette expression following transfection with act5c-GAL4 and UAS-ketteMyc or act5c-GAL4 and UAS-ketteMyr.

View larger version (33K): [in a new window]   Fig. 2. Kette binds to actin. (A) Soluble Kette protein was prepared as a maltose-binding-protein (MBP) fusion and stayed in the supernatant (S) when centrifuged at 200,000 g for 30 minutes. By contrast, F-actin was found in the pellet (P). When soluble Kette is added to F-actin, about 50% of Kette associates with the F-actin. Proteins were separated by SDS-PAGE followed by Coomassie blue staining. (B) MBP does not bind to F-actin. (C) Kette interacts with G-actin. Four clones of highly related actin sequences were identified in a yeast two hybrid screen. Sequence analyses indicate that interaction with Kette occurs at the C terminus.

View larger version (93K): [in a new window]   Fig. 3. Subcellular localization of endogenous Kette. (A,B) Primary tissue culture cells of Drosophila embryos were plated on fibronectin-coated cover slips and were allowed to differentiate over night. (C,D) Schneider S2 cells. Endogenous Kette protein was detected using anti-Kette antisera in a dilution of 1/1000. Anti-HRP antibodies recognize a carbohydrate present on all neuronal membranes. Anti-phosphotyrosine antibodies were used to detect focal contact sites. Phalloidin staining was used to visualize the F-actin cytoskeleton. A-C are projections of several confocal sections, D shows a single focal plane. Scale bar: 5 µm. (A) In neurons, Kette is expressed in an often punctuated pattern throughout the cell and can be detected in dendrites as well as axons. Higher levels of Kette are found at sites where the axon turns or branches (arrowheads). (B) Muscle cells are characterized by a highly regular F-actin cytoskeleton. Kette and F-actin expression largely overlap in these cells. (C) Schneider S2 cells endogenously express Kette. The majority of Kette is found in the perinuclear region (star). Small amounts of Kette are recruited to the leading edge of lamellipodia-like cell processes (arrowheads). Several Kette rich spokes extend from the nucleus to the membrane with higher levels of Kette at their end points close to the cell membrane (arrows). F-actin largely follows the Kette localization and can be detected surrounding the nucleus and in spokes extending to the membrane. In addition, a subcortical F-actin mesh can be detected in the lamellipodia-like structures just underneath Kette. (D) Kette expression as in C. Note the punctate appearance of Kette at the membrane. The distribution of tyrosine phosphorylated proteins frequently overlaps with Kette expression. Merged images are shown on the right.

View larger version (75K): [in a new window]   Fig. 5. Kette regulates F-actin organization. Kette expression (top), the F-actin cytoskeleton (middle) and the merge (bottom) are shown in Schneider S2 cells using anti-Kette antisera. All images represent stacks of confocal sections. (A) In wild-type cells, endogenous Kette protein largely colocalizes with F-actin. (B) Disruption of Kette expression by RNA interference leads to a concomitant disruption of the F-actin cytoskeleton. Forty-eight hours after treatment with kette dsRNA, aggregates of F-actin are found surrounding the nucleus (arrowhead). (C) Depletion of Scar/Wave by treatment with scar/wave dsRNA leads to a marked reduction in F-actin formation and alterations in cell morphology. Interestingly, in the absence of Wave, Kette appears to be uniformly distributed throughout the cell. (D) After RNAi for both kette and scar/wave, the formation of F-actin is not further reduced. (E) Overexpression of high levels of a Myc-tagged Kette protein does not lead to any changes in the organization of the F-actin cytoskeleton. The high dilution of the anti-Kette antiserum (1:50,000 compared with 1:2000 in A does not allow the detection of the endogenous Kette protein). (E) After expression of a membrane-tethered Kette protein, the F-actin cytoskeleton is rearranged. Large clumps of F-actin can be detected close to the membrane at sites that also show high levels of Kette expression. Scale bar: 5 µm.

View larger version (97K): [in a new window]   Fig. 6. kette antagonizes scar/wave function. Frontal views of embryonic nerve cords of stage 16 embryos. Axon tracts are labeled using Mab BP102 and HRP immunohistochemistry (brown), midline glia cells are labeled in blue (enhancer trap insertion AA142). (A) In a wild-type nerve cord, two commissures are found in each neuromere (anterior commissure, ac; posterior commissure, pc). They are clearly separated by the midline glia (star). (B) In homozygous mutant ketteC3-20 embryos the segmental commissures are not separated into distinct axon bundles and instead appear fused. (C) Removal of one copy of the scar/wave gene in a homozygous mutant ketteC3-20 embryo significantly restores CNS development and commissures are recognizable as distinct axon bundles. (D) Homozygous mutant scar/wave embryos display no mutant CNS phenotype. (E) Reduction of wasp function in a kette mutant embryo (wasp kette double mutant) does not modify the mutant kette phenotype. (F) Expression of a membrane-tethered Kette protein can rescue the kette mutant phenotype. A rho-GAL/UAS-ketteMyr; ketteC3-20/ketteC3-20 embryo is shown. Scale bar: 30 µm.

View larger version (124K): [in a new window]   Fig. 7. Kette regulates F-actin formation in vivo. (A-D) Top row, dorsal view of a Drosophila notum. Bottom row, higher magnification showing the morphology of microchaete and epidermal hairs (scale bar: 10 µm). (A) Wild-type flies are characterized by an ordered array of macro- and microchaete, which are normally thin, straight and with a pointed end (E). All epidermal cells generate a small hair. (B) After expression of three copies of a UAS-KetteMyr transgene in the scabrous pattern, bristle morphology is severely disrupted. KetteMyr expression results in shorter, branched and thicker bristles (G). In addition, epidermal cells generate more than one hair (arrow). The area boxed in red is shown enlarged. (C) After expression of two copies the UAS-KetteMyr transgene in the scabrous pattern results in a weaker phenotype; however, bristles are still forked and shorter (star and F) and epidermal cells develop more than one hair (arrow). The area boxed in black is shown enlarged. (D) Same genetic background as in C but lacking one copy of the wasp gene. The bristle phenotype evoked by KetteMyr expression is suppressed. (H) During pupal development, bristle morphology is prefigured by an apical F-actin extension. (I) After KetteMyr expression (three copies), F-actin formation is initiated in a broad region of the apical cell surface. In addition, the F-actin at the cell boundary appears to have a fuzzier organization after KetteMyr expression. Scale bars: 100 µm in A-D; 2 µm in E-G; 10 µm in H,I.

View larger version (46K): [in a new window]   Fig. 8. Abi binds Kette and Wasp. The yeast two hybrid system was used to determine the interaction between Kette and Wasp. The indicated constructs were tested for their ability to induce three distinct selection genes (-Ade, -His and -Gal reporters). The interactions of Kette and Abi and Wasp and Abi reconstitute functional GAL4 proteins, which enable yeast cells (AH109 strain) to grow on plates lacking adenine and histidine (Ade-, His-). In addition, these interactions activated -Gal expression as demonstrated by the blue color of the colonies. Yeast cells co-transformed with the Gal4 fusions pGAD-Kette and pGBK-Kette; pGAD-Kette and pGBK-Wasp failed to grow on selection plates.

View larger version (24K): [in a new window]   Fig. 9. Model of Kette function. Kette negatively regulates the activity of Wave in the cytosol but can activate Wasp at the membrane via Abi. Known interacting proteins are indicated. Kette also binds to F-actin, which helps to keep the Wave complex in a good position to stimulate formation of a meshed F-actin network. For further details see text.

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