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To corroborate that clathrin did not participate in W13-induced tubule formation, clathrin expression was inhibited by si RNA knockdown (Fig. Figure 1f shows that clathrin downregulation did not modify the extend of W13-induced tubules and, in agreement with the inhibition of recycling, W13 treatment accumulated transferrin in EEs vesicles at the cell periphery in contrast to its perinuclear localization observed in control cells.
These results indicate that induced tubular endocytic membrane structures are a cellular port of entry important for β1-integrin internalization in a CIE pathway.
In comparison with CDE pathways, the morphological features of membrane carriers generated by CIE pathways range from small vesicles to membrane tubular networks of different size and extension triggers the formation.
The same phenotype is generated by phosphatidylinositol 4-phosphate-5-kinase (PIP5K) overexpression or by treatment with the Ca M inhibitor N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13), which increase PI(4,5)P in the initiation of endocytic events is determined by its ability to bind and recruit several membrane-bending proteins such as dynamin or BAR-domain containing proteins, but also by its role in actin dynamics at the cell surface.
The images were acquired with a confocal microscope (Leica TCS SP5) and magnification insets show the presence of PI(4,5)P as in (c) (bars, 10 µm).
(e) In Cherry-Mem expressing cells, the percentage of cells presenting tubules and the number of tubules per cell were determined in different experimental conditions: W13 (20 min, 4.5 µg/ml), di C8-PI(4,5)P were determined by immunofluorescence performed as in (a).
This study provides new insights into Rac1 functioning on plasma membrane dynamics combining phosphatidylinositides and cytoskeleton regulation..
Mean values ± standard error of the mean (SEM) from three independent experiments are shown.
Actually, Rac1 could control tubule outcomes by regulating PI(4,5)P levels trigger dynamin-dependent endocytic tubules formation and enhance β1-integrin internalization, and that this process can be neutralized by Rac1 activation.
We show that Rac1 regulates PM endocytic tubule formation by controlling PI(4,5)P levels, actin dynamics and myosin activation through activation of PLC, cortactin and ROCK1, respectively.
1b), those cells that contained extensive tubulation (W13 treated) showed low β1-integrin labeling in EEs (Fig. Considering the presence in tubules as internalized molecules, cells exhibiting tubules showed increase in the total internalized β1-integrin after treatment with W13 at different time points (5, 10 and 20 min) compared with control cells (Fig. In these settings, the effect of W13-induced tubules on transferrin internalization, a well-established cargo of the CDE route, was also analyzed (Fig. Transferrin was not observed in W13-tubules and increased transferrin internalization was observed only at 20 minutes in W13-induced tubules compared to control cells, which could be explained by the previously reported effect of W13 inhibiting sorting from early endosomes and consequently transferrin recycling that at later time points contributes to the uptake measurements.
Likewise, this could be the reason for increased β1-integrin internalization at later time points in W13-treated cells not presenting tubules.