We demonstrate a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and Xenon arc lamp illumination. Our Bayesian analysis of blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores which may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame, and unifies the analysis of localization from blinking and bleaching events. By modeling the entire dataset we are able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allows us to reveal the nanoscale dynamics of podosome formation and dissociation with a resolution of 50 nm on a four second timescale.

mDia1, a Rho effector, belongs to the Formin family of proteins, which shares the conserved tandem FH1-FH2 unit structure. Formins including mDia1 accelerate actin nucleation while interacting with actin filament fast-growing ends. Here our single-molecule imaging revealed fast directional movement of mDia1 FH1-FH2 for tens of microns in living cells. The movement of mDia1 FH1-FH2 was blocked by actin-perturbing drugs, and the speed of mDia1 FH1-FH2 movement appeared to correlate with actin elongation rates. In vitro, mDia1 FH1-FH2 associated persistently with the growing actin barbed end. mDia1 probably moves processively along the growing end of actin filaments in cells, and Formins may be a molecular motility machinery that is independent from motor proteins.

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.Cell migration is indispensable in biological processes such as development, inflammation, wound healing, and tumor metastasis. Migrating cells polarize by extending protrusions at the front and retracting the tail at the rear, and make adhesions to extracellular matrix (ECM) to stabilize the forward protrusion (36). Adhesions to ECM are then used as sites to pull the cell body forward and are subsequently disassembled as the cell moves over them. This cycle of events enables cells to migrate to their destination. The actin cytoskeleton and microtubules (MTs) work critically in these events. Actin polymerization at the leading edge drives membrane protrusion, the association of the actin cytoskeleton with integrins regulates binding of the integrins to ECM, and the actin bundles within the body generate tension to pull the cell body forward and retract the tail. MTs are also polarized in migrating cells and are essential for the directed migration of many cell types (36, 37). However, how these cytoskeletons are regulated in migrating cells and work for cell polarization and adhesion turnover remains largely unknown.The Rho GTPases, including Rho, Rac, and Cdc42, work as molecular switches in cell morphogenesis by inducing specific types of actin cytoskeleton and by locally regulating MT dynamics. Accumulating evidence suggests that Cdc42 regulates cell polarity and Rac works in membrane protrusion of migrating cells. Indeed, Cdc42 is active at the cell front (17, 30), and disruption of Cdc42 function impairs directionality of migration in many cell types (1, 32). One well-characterized action of Cdc42 in cell polarity is to orient the MT organizing center (MTOC) in the front of the nucl...