JCS006049 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Different means of cell fixation and mDia2 localization reveal that mDia2 is in both a soluble and a filamentous-vesicular pool. (A) Paraformaldehyde or (B) methanol fixation and immunolocalization. (C-D) GFP-mDia2 fusion proteins expressed in PtK1 cells and imaged live by either spinning disk confocal (SDC; C) or total internal reflection fluorescence (TIRF; D) microscopy. (E) A PtK1 cell microinjected with an mDia2 antibody and fixed using paraformaldehyde, followed by immunolocalization of the injected antibody.

• Supplemental Figure S2 -

  Fig. S2. mDia2 antibody specifically inhibits mDia2 FH1FH2-mediated actin polymerization. Effect of mDia2 FH2 antibody on F-actin assembly. Pyrene-actin assembly assays containing 4 μM monomeric actin (5% pyrene-labeled), 2.5 nM of purified mDia2 FH1-FH2-C (red) or mDia1 FH1-FH2-C (blue), and the indicated μM concentrations of anti-mDia2 FH2 antibody.

• Movie 1 -

  Movie 1. TIR-FRAP reveals the kinetics of turnover of cortical actin in PtK1 cells. Control cell injected with X-Rhodamine-labeled actin, and imaged by TIR-FSM. X-Rhodamine actin is bleached at t=0 seconds. Subsequent images show fluorescence recovery.

• Movie 2 -

  Movie 2. mDia2 inhibition decreases the segregation of distinct actin kinematic behaviors in the lamellipodium and lamella. Control and mDia2 antibody-injected cells were injected with X-Rhodamine actin and FSM was performed on a spinning disk confocal microscope. The control cell shows well defined boundaries between different regions of distinct actin kinematics, whereas in the mDia2-inhibited cell, this segregation is less clear.

• Movie 3 -

  Movie 3. mDia2 inhibition alters the distribution and turnover of paxillin-containing focal adhesions in PtK1 cells. Control and mDia2 antibody-injected cells expressing GFP-paxillin were imaged on a spinning disk confocal microscope. The control cell shows the classic formation of focal adhesions behind the leading edge, and disassembly of older focal adhesions as the cell edge advances, producing a distribution of focal adhesions in a band behind the leading edge. In the mDia2-inhibited cell, focal adhesions still form behind the leading edge, but fail to disassemble, creating a distribution of focal adhesions throughout the cell.