Stx was the first lipid-binding ligand shown to induce its own uptake through a clathrin dependent pathway 
. Our data provide evidence that Stx/StxB, upon binding to its glycolipid receptor at the cell surface, is able to increase the formation of endocytic clathrin-coated structures directly detected by electron microscopy and indirectly by the appearance of AP-2 spots at the plasma membrane. Furthermore, data obtained using live cell spinning disk confocal imaging indicates that the new coated structures tend to have longer lifetimes and higher maximum fluorescence intensities, presumably reflecting slightly larger coats. The tyrosine kinase Syk seems to be important for this activation, although the mechanism responsible for this process remains to be established.
We first used electron microscopy to demonstrate an increase in the number of clathrin-coated pits in HeLa or HEp-2 cells exposed to Stx. We then confirmed this finding for HeLa cells exposed to StxB by using live-cell spinning disk confocal imaging. We note that our data might underestimate the full extent of the effect because we did not include in the analysis clathrin/AP-2 coated plaques, corresponding to endocytic structures lasting more than 2–3 min 
. We noticed differences in the response of given cells that we interpret to be due to the existence of cells that poorly bind Stx, probably because of differences in the expression level of the Stx glycosphingolipid receptor Gb3.
It is presently unclear whether the increase of AP-2 structures mediated by Stx is explained by activation of initiation events leading to an increase in the number of the de novo formed coated pits or whether Stx activation stabilizes abortive pits allowing them to become fully formed coated vesicles 
. Stx can induce non-apoptotic signaling cascades in cells, but the mechanism behind this rapid signaling, induced by binding of its B-moiety to Gb3, remains to be established 
. It has been proposed that the lipid tail of glycosphingolipids can interact with the inner membrane leaflet, and signaling may also be mediated through a membrane protein interacting with the Stx/Gb3-complex. It has recently been shown that the extra-cellular domain of the trans-membrane receptor Fas interacts strongly with Gb3 through a glycolipid-binding motif 
. This motif is important for Fas-induced apoptotic and non-apoptotic signaling as well as clathrin-mediated internalization of Fas 
. Moreover, Stx has been shown to interact with two non-identified, possibly signaling proteins at the cell surface 
. We have previously demonstrated that increased concentrations of Stx, as opposed to StxB, lead to higher uptake of TAG/biotin-labeled Stx 
. Thus, the A-moiety seems to be required for the concentration-dependent toxin uptake, yet StxB is sufficient for signaling and increased clathrin-coated pit formation.
Our data show that the Stx-induced formation of the AP-2-containing clathrin-coated structures reached its maximum value after about 10–13 min of incubation with the toxin. This observation coincides with the timing of clathrin heavy chain phosphorylation induced by Stx that peaks at around 10–15 min after toxin addition 
. Of note, activation of Syk by Stx is faster and can be detected already 2.5 min after addition of Stx 
. Clathrin and Syk can associate with each other, and they form a complex upon induction by Stx 
. Syk is not only important for Stx endocytosis 
but it also seems to be involved in the uptake of human rhinovirus 
; interestingly, virus binding induces Syk recruitment to the plasma membrane and co-association with clathrin. We found that inhibition of Syk activity by piceatannol prevented the increase in the number of clathrin-coated structures that formed upon addition of StxB. Although these observations clearly indicate a connection between Syk activation and regulation of the clathrin endocytic pathway, the detailed mechanism responsible for this process remains to be determined. These observations also seem to rule out a simple model of clathrin coat activation that could be based on changes in the physical properties of the lipid bilayer at the site of coat formation due to local clustering of glycosphingolipids induced by binding to the pentameric StxB.