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First published online 6 February 2007
doi: 10.1242/jcs.03378

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Tau impacts on growth-factor-stimulated actin remodeling

Vandana M. Sharma, Joel M. Litersky, Kiran Bhaskar^* and Gloria Lee

Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

View larger version (59K): [in this window] [in a new window]   Fig. 1. Tau increases SFK activity. (A-D) Tyrosine phosphorylation of tubulin in the presence of tau and Fyn or Src. (A) E. coli tau and/or Fyn were incubated and added to microtubules. After centrifugation, the supernatant (S) and microtubule pellet (P) were probed with anti-phosphotyrosine (see Materials and Methods). While microtubules were phosphorylated by Fyn (lane 4), tau increased the level of phosphorylation (lane 8). Autorad was deliberately overexposed to show tyrosine phosphorylated tau in lanes 5 and 8 (asterisk). (B) N-terminal fragment of tau and Fyn were incubated, added to microtubules, and probed as described in A. (C) Microtubules isolated from COS7 cells transfected with full-length tau and/or Fyn were probed with anti-phosphotyrosine (top panel) or anti-tubulin (bottom panel). (D) Tau and Src were incubated, added to microtubules, and probed as described in A. (E) Tau activates autophosphorylation of Src. 0.2 or 2 µM tau (lanes 1 and 2, respectively) was incubated with Src and probed for active Src, total Src, and tau as described in Materials and Methods.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Tau expression causes changes in actin reorganization following PDGF stimulation. 3T3 cells transiently transfected with tau were stimulated with PDGF for 10 minutes or 7 hours, fixed and labeled with phalloidin-Alexa594. Panels A-C show the actin morphology of non-transfected control cells following PDGF treatment as indicated. Panels D-F show the actin morphology in tau-expressing cells similarly treated. Tau-transfected cells were identified by labeling with the polyclonal tau antibody CR (G-I). (J) Quantification of cells showing PDGF-induced actin fiber breakdown in the presence and absence of tau. Percentage of non-transfected cells (open bars) and tau-transfected cells (black bars) showing actin stress fiber breakdown was determined after either 10 minutes or 7 hours PDGF treatment as described in Materials and Methods. (K-L) Expression of a constitutively active form of Src also disorganized the actin cytoskeleton. Labeling for activated Src (phospho-Y416) (K) and phalloidin (L) are shown. Bar in A, 10 µm (applies to panels A, B, D-I). Bar in C, 10 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Taxol treatment of 3T3 cells does not affect PDGF-induced actin reorganization. 3T3 cells treated with 100 nM taxol were stimulated with PDGF and double-labeled with Phalloidin-Alexa594 (A,C,E) and anti-acetylated tubulin (B,D,F). Bar, 10 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 4. Loss of microtubule-binding activity does not alter the effects of tau on PDGF-induced actin reorganization. 3T3 cells were transfected with S262D/S356D mutant tau, stimulated with PDGF, and double-labeled as described in Fig. 2 (phalloidin-Alexa594 in A,B,C and anti-tau in D,E,F). The mutated tau exhibited a diffuse cytoplasmic distribution due to its loss of microtubule binding. Bar, 10 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 5. Activated Src distribution in tau-transfected 3T3 cells following PDGF stimulation. 3T3 cells were transfected with tau, stimulated with PDGF, and double-labeled with anti-Src (phospho-Y416) (A-F) and anti-tau (G-I). Quiescent cells showed activated Src located in focal adhesions (Fincham and Frame, 1998) (A,D). Upon stimulation with PDGF, activated Src localized to the periphery in a punctate pattern (B,E). At 7 hours, while the Src distribution reverted to the focal adhesions in non-transfected cells (C), in tau-expressing cells, much activated Src was still localized at the periphery (F). The tau distribution (G-I) was non-fibrillar owing to the fixation conditions required for labeling with the activated Src antibody. Bar, 10 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Effect of Src inhibition on tau-induced alterations in actin reorganization. The top panel shows a schematic diagram of the different protocols of PP2 treatment. Micrographs show 3T3 cells transfected with tau, treated with 10 µM PP2 and PDGF, and double-labeled with anti-tau (A,C,E) and phalloidin-Alexa594 (B,D,F). Cells received either PP2 and PDGF simultaneously (A,B), PP2 45 minutes in advance of PDGF (C,D), or PP2 5 hours after PDGF addition (E,F) as described in the Results and shown in the schematic above. Bar, 10 µm. (G,H) Quantification of cells showing PDGF-induced actin fiber breakdown in the presence of PP2. Percentage of non-transfected and tau-transfected cells showing actin stress fiber breakdown in the presence of Src inhibition was determined using three protocols of PP2 treatment. PP2+PDGF indicates simultaneous addition of PP2 and PDGF with actin fiber breakdown assessed 10 minutes after addition (protocol 1). PP2PDGF indicates 45 minutes of PP2 treatment in advance of PDGF addition, with actin fiber breakdown assessed 10 minutes after PDGF addition (protocol 2). PDGFPP2 indicates 5 hours PDGF incubation in advance of PP2 addition, with actin fiber breakdown assessed after 30 minutes PP2 treatment (protocol 3). Data for untreated cells were reproduced from Fig. 2J for comparison. (I) Effect of the tau phosphomimicking mutant on actin stress fiber breakdown. The percentage of cells transfected with the tau T231D/S235D mutant showing actin stress fiber breakdown was determined after either 10 minutes or 7 hours PDGF treatment. Significant differences are *P<0.05 and **P<0.001, respectively, between transfected and non-transfected cell percentages. Non-transfected cells are indicated with open bars, tau-transfected cells with black (G,H) or striped (I) bars.