TRPV4 Regulates Cytoskeleton Figure 4. TRPV4 localizes in the growth cone and regulates axonal motility as activation of TRPV4 results in microtubule disassembly. a. Shown are the confocal time series images of live F11 cell expressing TRPV4-GFP (green) and RFP-Actin (red). Fluorescence images were superimposed on the DIC images. Addition of 4aPDD (1 mM) results in growth cone retraction of the transfected cell (T) but not from the non- transfected (NT) cells. b. Prolonged activation of endogenous TRPV4 reduces neurite outgrowth. Shown are the images of cultured DRG neurons stained for IB4 (red) and bIII tubulin (green). IB4-positive neurons extend their neurites in control condition (upper panel). Majority of the IB4-positive neurons do not produce any neurite when 4aPDD at low dose (0.1 mM) was applied for 36 hours (middle panel). An enlarged view of an IB4-positive and an IB4-negative neuron is shown in the lower panel. Note that the majority of the IB4-negative neurons remain unaffected even in the presence of 4aPDD. c. CHO-KI-TRPV4 cells that express low level of TRPV4 or CHO-KI-Mock cells that do not express TRPV4 were activated with 4aPDD (1 mM). After activation, cells were extracted by detergent in isotonic buffer and fixed subsequently by PFA. Cells were immunostained for actin (green) and tubulin (red). CHO-KI-TRPV4 cells loose all the peripheral microtubules but retain filamentous actin after activation and extraction. The stable MTOC regions are marked with arrows. In contrast, CHO-KI-Mock cells remain unaffected. Intensity of the microtubule is shown (red and blue indicate highest and lowest intensity respectively). doi:10.1371/journal.pone.0011654.g004 PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11654