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.