Supplementary Materials Supplemental Material supp_201_6_903__index. uterine AC breaches the cellar membrane separating the uterine and vulval epithelium to determine the original connection between these cells. The timing of AC invasion can be highly stereotyped and it is firmly coordinated using the division from the root vulval cells (Fig. S1 A). Both integrin heterodimer INA-1/PAT-3 as well as the netrin receptor UNC-40 (DCC) localize towards the cell membrane from the AC in touch with the cellar membrane and are thought to promote invasion by regulating F-actin polarized toward the ACCbasement membrane interface (Hagedorn et al., 2009; Ziel et al., 2009). How these pathways specifically mediate F-actin dynamics and facilitate basement membrane breaching and transmigration is unclear. By developing methods for time-lapse microscopy of AC invasion, we identify here highly dynamic, integrin-dependent, F-actinCbased structures that breach the basement membrane. These protrusive, F-actinCrich foci share molecular composition and activity with invadopodia, invasive structures identified in transformed and highly invasive cancer cells in vitro (David-Pfeuty and Singer, 1980; Chen, 1989; Murphy and Courtneidge, 2011). Many invadopodia form and turn over within the AC before invasion, but usually only one penetrates the basement membrane and then gives rise to a stable order LGK-974 protrusion that invades the vulval tissue. We find that the netrin receptor UNC-40 (DCC) is highly enriched at sites of basement membrane breach and within the invasive protrusion. Animals lacking UNC-40 fail to form an invasive protrusion and have persistent invadopodia, multiple basement membrane breaches, and inefficient basement membrane removal. Using optical highlighting of basement membrane components, we show that this UNC-40Cdependent invasive protrusion accelerates basement membrane removal in part by physically displacing this barrier. Together, these studies reveal an UNC-40 (DCC)Cdependent mechanism that ensures focused invasion at nascent sites of basement FLJ34064 membrane breach. Outcomes Active F-actinCbased AC-invadopodia breach the cellar membrane To research the way the cellar can be crossed from order LGK-974 the AC membrane, order LGK-974 we developed strategies combining non-toxic anesthesia, multidimensional time-lapse microscopy, and quantitative picture analysis to see the intrusive cellCbasement membrane user interface instantly (Fig. 1 A and Video 1). Actin dynamics and cellar membrane integrity had been concurrently visualized using pets expressing AC-specific F-actin probes (or = 8,182 constructions from 10 pets) and a median duration of 45 s (which range from 15 to 930 s). Before invasion, nascent constructions formed at a reliable price of 2.8 0.4 set ups each and every minute and there have been normally 11 3 set ups present (Fig. S1 E). Open up in another window Shape 1. F-actinCrich AC-invadopodia breach the cellar membrane. (A) A schematic diagram depicts both perspectives useful for time-lapse imaging of AC invasion. (best) Wild-type pets lay on the side, permitting AC invasion to laterally become imaged. The cellar membrane can be visualized with laminin::GFP in magenta as well as the AC-specific F-actin probe can be demonstrated in order LGK-974 green and overlaid on differential disturbance contrast pictures. (bottom level) mutant pets orient arbitrarily, permitting ventral imaging. Confocal pieces through the ACCbasement membrane user interface are demonstrated at magnification of two in accordance with best panels. Orientation can be indicated in every sections. (B) Ventral look at time series displays dynamic areas of F-actinCrich invadopodia in the ACCbasement membrane user interface. To demonstrate the rapid price of turnover, coloured spots had been assigned to fresh F-actin constructions at 2-min intervals (discover Fig. S1 for comprehensive info on size, life time, rate of development, and amount of invadopodia as time passes). The behavior of the F-actin constructions was identical when visualized with Lifeact::GFP so when worms had been immobilized by compression (Fig. S1). (C) Before breaching, invadopodia (middle, arrow) may actually depress the cellar membrane (correct, arrow). (D) At the time of basement membrane breach an invadopodium (middle, arrow) occupies the site of breach (right, arrow). (E) A ventral view time series shows an AC-invadopodium (middle row, arrows) presaging and then occupying a visible basement membrane breach (bottom, arrows; similar results were observed in 8/8 animals). Bars, order LGK-974 5 m. F-actinCrich, matrix-degrading membrane structures called invadopodia have been identified in a variety of malignant cancer cell lines, as well as transformed fibroblasts and zebrafish epithelial cells (Linder et al., 2011; Murphy and Courtneidge, 2011;.