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Figure 1


Fig. 1. PtK1 cells express mDia2, which localizes in the lamella and cell center and not to the lamellipodium. Whole cell lysates from HEK 293T cells expressing either YFP-fused mDia1 (A) or Myc-tagged mDia2 (B), as well as HeLa and Ptk1 cell lysates (A-C) were prepared. Lysates were immunoblotted with antibodies directed against mDia1 (A), mDia2 (B), or mDia3 (C). (D) Fluorescent phalloidin staining of F-actin and immunofluorescence of tubulin and mDia2. In the merged image, tubulin is blue, mDia2 is green, and F-actin is red. mDia2 partially colocalizes with microtubules in the lamella and is excluded from near the cell edge. The cell edge as determined from phase-contrast images (not shown) is outlined in white in this and subsequent images. (E) Immunofluorescent staining for Arp3 and mDia2 in PtK1 cells. In the merged image, Arp3 is in red, and mDia2 is in green. Arp3 concentrates in a thin band along the cell edge in the lamellipodium where mDia2 is depleted. (F) Immunofluorescent staining for myosin II regulatory light chain phosphorylated at Ser-19 (pMLC) and mDia2. Merged image shows pMLC in red and mDia2 in green. mDia2 is present in the lamella where pMLC is concentrated. (G) Paxillin and mDia2 immunofluorescence, paxillin in red, mDia2 is in green. mDia2 and paxillin do not colocalize.





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