Supplementary Materials Supplemental Materials supp_28_14_1924__index. movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP Cilazapril monohydrate structures symbolize a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration. INTRODUCTION Cell movement requires the spatial control of transmission transduction, including cell polarity mechanisms that move proteins to specific intracellular locations (Huttenlocher, 2005 ; McCaffrey and Macara, 2012 ). During cell locomotion, cells must coordinate the formation of membrane protrusions and attachments to extracellular matrix at the front, with membrane retraction and disassembly of attachments at the rear. Much is known about events at the leading edge, where actin polymerization via Rac, Cdc42, WASP/WAVE, and Arp2/3 form lamellipodia and membrane protrusions, which promote focal contact attachments to extracellular matrix and mediate forward movement (Ridley 0.01. The values were calculated using standard Rabbit Polyclonal to CHML two-tailed Students test. The term polarized in this figure does not distinguish between rear and front polarity. WRAMP structures were quantified by immunostaining of endogenous MCAM and myosin IIB and phalloidin staining of F-actin. Treatment of cells for 30 min with Wnt5a significantly enhanced the percentage of cells showing WRAMP structures, which increased by 2.5- to 3-fold as measured by polarized localization of MCAM (Determine 1C). Typically, WRAMP structures form within 15 min, but quantified at a single time point, they appear in only 24% of WM239A and 15% of Cilazapril monohydrate A375 cells. This is explained by their transient nature; they assemble dynamically, followed by disassembly. When measured over a period of 0?2 h, 80% of cells formed WRAMP structures (unpublished data). Approximately 20% of WM239A cells and 12% of A375 cells showed F-actin polarized along with MCAM after Wnt5a treatment (Physique 1D). Therefore F-actin was present in 80% of WRAMP structures based on polarized MCAM. We also found myosin IIB polarized at WRAMP structures in 50% of cases (Physique 1E). F-actin was present in almost all of the WRAMP structures with myosin IIB (Physique 1F). Thus WRAMP structures were characterized by strong association between polarized MCAM, F-actin, and myosin IIB, forming with coordinately increased frequency in Cilazapril monohydrate response to Wnt5a. WRAMP structure formation entails coordinated movement of MCAM, F-actin, and myosin IIB Confocal live cell imaging was used to examine the order of MCAM, F-actin, and myosin IIB recruitment into WRAMP structures. In both WM239a and A375 cells, MCAMCgreen fluorescent protein (GFP) polarized dynamically to form WRAMP structures and was usually followed by membrane retraction (Physique 2, A and B). Cells were also cotransfected with MCAM-GFP and mCherry in controls, which confirmed that this polarized localization of MCAM-GFP was not an artifact caused by variations in cell volume or membrane thickness (Supplemental Physique S1). We then examined 100 cells cotransfected with MCAM-GFP and LifeAct-mCherry, a peptide that binds and labels F-actin. In each case, the accumulation of F-actin into WRAMP structures was synchronous with the polarization of MCAM-GFP (Physique 2, A and B). WM239a cells migrated in a manner that reflected distributing and adhesiveness reminiscent of mesenchymal cell movement, whereas A375 cells migrated with more-rounded morphologies. Nevertheless, the temporal dynamics of F-actin and MCAM-GFP in forming WRAMP structures were tightly coordinated in each cell type. Open in a separate windows FIGURE 2: Dynamic movement of WRAMP structures, followed by membrane retraction. Frames from confocal live-cell imaging experiments of (A) WM239a and (B) A375 cells cotransfected with MCAM-GFP and LifeAct-mCherry and (C) WM239a and (D) A375 cells cotransfected with MCAM-GFP and myosin IIB-N18-mCherry. Supplemental Movies S2CS5 (corresponding to ACD, respectively) show coordinated movement of MCAM, F-actin, and myosin IIB. White dot indicates starting position. Scale bars, 10 m; occasions in hours:moments. Controls for this experiment with MCAM-GFP plus mCherry.