Pitstop 2 Is a Potent Inhibitor of Clathrin-Independent Endocytosis

Abstract

Clathrin independent endocytosis (CIE) is a form of endocytosis present in all cells that mediates the entry of nutrients, macromolecules and membrane proteins into cells. When compared to clathrin-dependent endocytosis (CDE), however, much less is known about the machinery involved in forming CIE endosomes. One way to distinguish CIE from CDE has been to deplete cells of coat proteins involved in CDE such as clathrin or the dynamin GTPase, leading to a block of CDE but not CIE. A drawback of such genetic manipulations is that depletion of proteins important for mediating CDE over a period of days can have complex indirect effects on cellular function. The identification of chemical compounds that specifically and rapidly block CDE or CIE would facilitate the determination of whether a process involved CDE or CIE. To date, all of those compounds have targeted CDE. Dynasore and the dynoles specifically target and block dynamin activity thus inhibiting CDE but not most forms of CIE. Recently, a new compound called pitstop 2 was identified as an inhibitor of the interaction of amphiphysin with the amino terminal domain of clathrin, and shown to inhibit CDE in cells. Here we show that pitstop 2 is also a potent inhibitor of CIE. The effects of pitstop 2 are not restricted to inhibition of clathrin since knockdown of clathrin fails to rescue the inhibition of endocytosis of CIE proteins by the drug. Thus pitstop 2 has additional cellular targets besides the amino terminal domain of clathrin and thus cannot be used to distinguish CIE from CDE.

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…Our data question the conclusion of a previous study that the Pitstop-2 compound has an off target action in clathrin-independent endocytosis (39). Using the same cell type and conditions as reported in that study (and, thus, ruling out technical or cell type-specific differences), we show based on both RNAi against clathrin and AP-2( ) and two different Pitstop compounds as well as inactive analogs that MHCI endocytosis is indeed mediated by clathrin and AP-2 (Fig.…”

“…We note that in none of the previously published works by Donaldson and coworkers, including Ref. 39, is a direct quantitative comparison of control versus clathrin-or AP-2-depleted cells with respect to their ability to internalize MHCI presented. Instead, it was claimed based on overexpression of a supposedly GTP-locked dominant-negative Arf6 mutant that MHCI follows an Arf6-specific clathrin-independent endocytosis pathway (6,40,52).…”

“…Because Donaldson and co-workers (39) reported Pitstop-2-mediated inhibition of MHCI endocytosis, a process claimed to be clathrin-independent (40), with a dose dependence identical to that of the CME cargo transferrin, we started out by reinvestigating the role of clathrin and its major endocytic adaptor AP-2 in MHCI uptake. Antibodies against surface MHCI were allowed to internalize at physiological temperature for 30 min and visualized by confocal microscopy using previously established protocols and cell types (39). Application of Pitstop-2 potently inhibited MHCI endocytosis (Fig.…”

The architectural relationship of components controlling mast cell endocytosis

“…We also confirm that FceRI endocytosis by both routes is dependent upon dynamin (Fattakhova et al, 2006) and show that, although the CME pathway can be utilized by aggregated FceRI, it is unlikely to be the major route. FceRI internalization is blocked by Pitstop2, a reagent that has been recently shown to affect both pathways (Dutta et al, 2012;Lemmon and Traub, 2012).…”

“…This compound was originally described as a specific CME inhibitor, based upon its direct association with clathrin terminal domain (TD) and on in vitro evidence that it inhibits formation of the complex between the clathrin TD and amphiphysin. A recent article by Donaldson and colleagues indicates that Pitstop2 also blocks CIE, raising the possibility that this drug has other common or distinct targets in the endosome sorting process (Dutta et al, 2012).…”