Tail domains of myosin-1e regulate phosphatidylinositol signaling and F-actin polymerization at the ventral layer of podosomes.

During the podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3 dependent manner. Combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Over-expression of TH12, not TH12 KR2A impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome. Movie S1 Movie S1 Photo-conversion of cytosolic mEOS2-β-actin reveals the assembly process of F-actin at the ventral layer of the podosome in MEF-Src. Spot activation of 405nm laser is applied at the circled area away from podosome arc, and photo-converted cytosolic mEOS2-β-actin can freely diffuse with the cell. After photo-conversion, 561nm excitation is used to monitor the incorporation of photo-converted actin into different layers of the podosome by a spinning disk confocal microscope. At first, newly converted mEOS2-actin incorporate into the ventral layer (z= 0μm) and then gradually move to the upper layers of podosome. Scale bar represents 10μm. Movie S2 Movie S2 PI(3,4,5)P3 lipids are enriched at the podosome. PH-BTK and PH-PLCδ are used as specific probes to visualize the distribution of PI(3,4,5)P3 and PI(4,5)P2 lipids, respectively. During the podosome formation in MEF-Src, PH-BTK is enriched at the F-actin core in the podosome, whereas the level of PH-PLCδ is reduced. Scale bar represents 10μm. Movie S3 Movie S3 Fluorescence recovery after photobleaching (FRAP) of \ full-length Myo1e and various tail constructs at the podosome in MEF-Src. The recovery of full-length Myo1e is slow. Different from full-length Myo1e, TH1, TH2, and TH3 all have fast recoveries after photobleaching. TH12 and TH123, on the other hand, exhibit similar slow recoveries as full-length Myo1e. See Fig. S5C for the statistical information. Scale bar represents 10μm. Movie S4 Movie S4 The expression of TH12 suppresses the formation of podosome rosettes and arcs. F-actin polymerization at the podosome becomes short-lived and unstable in TH12-expressed MEF-Src. Scale bar represents 10μm. Movie S5 Movie S5 Dynamical assembly of podosomes in RAW 264.7 macrophage. Myo1e is localized at the F-actin core in each podosome. Scale bar represents 10μm. Movie S6 Movie S6 The expression of TH12 disrupts the podosome formation in RAW 264.7 macrophage. F-actin fails to assemble distinct podosomes. Scale bar represents 10μm.