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pxxx stage ( Fig. 2I). madd-2(tr103) did not suppress the invasion defect of fos-1a(ar105) mutants but rather caused a slightly enhancement, as all of 35 fos-1a(ar105); madd-2(tr103) double mutants examined had an intact BL ( Fig. 2J, p-valueTyrosine Kinase Inhibitor Library cost guidance rather than the invasiveness induced by FOS-1A. To gain further insight into madd-2 function, we examined AC shape and polarity using the mCherry::PLC��PH reporter, which binds to phosphatidylinositol-(4,5)-bis-phosphate (PIP2). Highest PIP2 levels are observed at the ventral, invasive plasma membrane, where actin polymerizes and AC protrusions are formed ( Fig. 3A, B) ( Luna and Hitt, 1992?and?Ziel et al., 2009). In contrast to the bell-shaped appearance of the AC in the wild-type and the round shape in unc-6(ev400) mutants, the AC in madd-2(tr103) single and madd-2(tr103); unc-6(ev400) double mutants adopted a rectangular shape with irregular borders ( Fig. VAV2 3A�CF, suppl. Fig. s1A). Using the PIP2 reporter to measure AC polarity, we found that in madd-2(tr103) and unc-6(ev400) single as well as in the double mutants the AC was less polarized than in the wild-type ( Fig. 3G, suppl. Fig. s1B). Thus, loss of madd-2 function did not fully restore AC polarity in the absence of UNC-6 signaling. We also investigated whether the loss of AC polarization in madd-2(tr103) mutants affects the direction of invasion by observing the localization of HIM-4::GFP, which the AC normally secretes only ventrally toward the BL ( Fig. 3H) ( Sherwood et al., 2005?and?Vogel and Hedgecock, 2001). In madd-2(tr103) mutants, around 8% of total HIM-4::GFP protein was secreted on the dorsal side of the AC ( Fig. 3I, suppl. Fig. s1C, D). Taken together, these results indicated that madd-2 is required to polarize the AC and selleck compound direct its invasive machinery ventrally toward the BL. Given the changes in AC shape and polarity, we hypothesized that the AC in madd-2(tr103) mutants might form ectopic protrusions, leading to an overall increased invasiveness. By observing the changes in AC shape with time-lapse microscopy, we found that the AC in madd-2(tr103) mutants was more dynamic than in the wild-type or in unc-6(ev400) single mutants, frequently forming ectopic protrusions on the dorsal or lateral plasma membranes ( Fig. 4A�CD, suppl. movies s1�C4). Quantification of the recordings using an unbiased computer algorithm to measure AC shape changes (see Materials and methods section) confirmed the increased variability in AC shape, both in madd-2(tr103) single and madd-2(tr103); unc-6(ev400) double mutants ( Fig. 4E). The formation of undirected, ectopic membrane protrusions could explain why loss of madd-2 function partially suppresses the basal lamina breaching defect of unc-6(ev400) mutants. The following is the supplementary material related to this article Video 1, Video 2, Video 3, Video 4 To view the video inline, enable JavaScript on your browser.