PKCζ Is Required for Microtubule‐Based Motility of Vesicles Containing the ntcp Transporter

Abstract

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na + -taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCz have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCz and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCz blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCz is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.

Kif5B and Kifc1 Interact and Are Required for Motility and Fission of Early Endocytic Vesicles in Mouse Liver

“…It is of interest that in contrast to results with antibody to Kifc1, overall motility of vesicles is reduced after incubation with antibody to Kif5B. This is in agreement with results observed in several previous studies (Brady et al, 1990;Martin et al, 1999;Ligon et al, 2004;Theiss et al, 2005;Sarkar et al, 2006), suggesting that Kif5B activity is dominant over other motor activities (Sarkar et al, 2006). The finding that fission of vesicles is reduced when either Kifc1 or Kif5B activity is inhibited by antibody also suggests that the opposing forces resulting from activity of both motors are required for fission to occur.…”

“…There was no evidence that the minus-end motility of these vesicles was due to substitution of dynein for Kifc2. Specifically, there was no inhibition of vesicle motility in the presence of 5 M vanadate (Figure 4), a treatment that inhibits dynein-mediated motility Sarkar et al, 2006), and there was no immunocolocalization of dynein with fluorescent ASOR-containing vesicles ( Figure 6A). Rather, as was found in studies of early endocytic vesicles from the rat, all motility in both wild-type and Kifc2 knockout mouse vesicles was inhibited by 1 mM AMP-PNP, an inhibitor of kinesin based motility (Vale et al, 1992;Bananis et al, 2000;Murray et al, 2000).…”

“…Alternatively, they could represent a different population of early/recycling endocytic vesicles such as those that contain the bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) (Sarkar et al, 2006). These ntcp-containing vesicles cycle to and from the basolateral plasma membrane of hepatocytes, by using Kif5B and dynein (Sarkar et al, 2006). Because these data indicated the presence of a minus-enddirected kinesin on mouse early endocytic vesicles and Kifc2 was absent, we considered the presence of other minus-end kinesins, of which only four others have been described previously (Yang et al, , 2001a(Yang et al, ,b, 2006Navolanic and Sperry, 2000;Noda et al, 2001;Miki et al, 2003;Yang and Sperry, 2003;Zhang and Sperry, 2004).…”

“…As processing of endocytic vesicles is dynamic (Bananis et al, 2004;Sarkar et al, 2006), it is possible that these vesicles had already passed a transition point toward late vesicles, which we have shown are associated with dynein (Bananis et al, 2004). Alternatively, they could represent a different population of early/recycling endocytic vesicles such as those that contain the bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) (Sarkar et al, 2006). These ntcp-containing vesicles cycle to and from the basolateral plasma membrane of hepatocytes, by using Kif5B and dynein (Sarkar et al, 2006).…”

A stimulus needed for the study of membrane traffic in hepatocytes†

“…Recent studies have shown that transporter-containing intracellular vesicles can move along microtubules, powered by specific members of the molecular motor families, dynein and kinesin. 6,7 Although exact mechanisms still need to be discovered, the direction of movement can be regulated by the activity of specific vesicle-associated kinases and rabs. Interestingly, the proteins and motors associated with these vesicles appear to differ, depending on the transporter.…”