supervision; D

supervision; D. YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity. mechano-sensitive regulator of gene expression (8, 11, 12, 23). Therefore, we asked whether LAMTOR-positive LE distribution and dynamics were mechano-sensitive and could act as a molecular link between integrins and YAP activation. First, we verified that YAP activation was mechano-sensitive in the preosteoblast cell line used for this study. YAP expression was mainly nuclear in cells cultured on fibronectin-coated polydimethylsiloxane (PDMS) medium and stiff hydrogels (10 and 30 kPa). Lowering compliance to 2 Transcrocetinate disodium kPa led to a significant YAP redistribution in the cytoplasm (Fig. 1, and and and and test and are representative of two independent experiments with 30 cells analyzed (***, 0.0001; test). test). and and 30 (two-tailed Transcrocetinate disodium unpaired Student’s test). 30 cells analyzed (two-tailed unpaired Bmpr1b Student’s test). osteogenic differentiation of preosteoblast cells (1f/f) that stably express scramble (sh-ctl) or shRNAs against p18/LAMTOR1 (sh-p18). Alkaline phosphatase ( 0.0001. Open in a separate window Figure 5. p18/LAMTOR-dependent SRC delivery to the plasma membrane controls YAP nuclear shuttling. test). test). test). Subcellular localization of YAP in sh-p18 preosteoblast cells and in sh-p18 cells that express constitutively active SRCYF (sh-p18+SRCYF). 0.0001; and and Fig. S1and 30 cells analyzed (two-tailed Transcrocetinate disodium unpaired Student’s test). 0.0001), 10C12 m ( 0.001) and 8C10 m ( 0.01). Due to the lack of an appropriate tagged p18/LAMTOR1 construct, rescued cells were not investigated in this experiment. test was used with = 20 cells/condition. *** 0.0001. 30 cells/condition; two-tailed unpaired Student’s test). test (= 15 cells/condition). and in Fig. 3in Fig. 3 20 cells/condition) were compared with the two-tailed unpaired Student’s test. 30 cells/condition) (two-tailed unpaired Student’s test). *, 0.01; **, 0.001; ***, 0.0001; and ((p18/LAMTOR1) overlapped with the (SRC-mCherry). The smaller tM2 value (overlapping with and in Fig. 5and Fig. S3and Fig. S3and peripheral) determines its function (catabolic anabolic) (13, 14, 16, 17). Our present work supports this idea by showing that FA-associated LEs promote YAP nuclear translocation, a well-known cell growth promoter. The LAMTOR complex, which was isolated from late endosomal detergentCresistant membranes, is involved in the regulation of LE dynamics and signaling (16, 18, 19). In agreement, we observed that p18/LAMTOR1 has a critical role in LE targeting to the Transcrocetinate disodium plasma membrane and FAs. Our data suggest Transcrocetinate disodium that this is independent of its signaling function in the mTORC1 pathway. It was reported that LAMTOR restricts LE distribution to the perinuclear area by inhibiting the Arl8/BORC complex (19). We propose that LAMTOR is also required for the peripheral delivery of LEs that are targeted to adhesive structures. Although in apparent contradiction, these discrepant observations may be explained by the methods used to assess LE distribution and/or by the different cell types used. Indeed, in previous reports, LE distribution was analyzed by quantifying LE markers from the nuclear barycenter or the microtubule-organizing center as the origin, without delimiting the cell borders. Here, we accurately delimited the cell borders and quantified the vesicle densities from this position. This is particularly important in cells that generate large lamellipodia, such as mesenchymal cells. Indeed, we observed that upon p18/LAMTOR1 silencing, LE density was reduced mostly within the lamellipodial region. One important LAMTOR function is to regulate mTORC1 activation. In agreement with previous reports, mTORC1 signaling appears to be dispensable for YAP nuclear translocation and also for.