Actin is already known to be important in endocytosis. But, in The Journal of Cell Biology, Drubin and colleagues now report new roles for actin and the HIP1R linker protein in the budding from the trans-Golgi network (TGN) of clathrin-coated vesicles (CCVs) that are bound for lysosomes.

Actin is involved in CCV formation at the plasma membrane and short actin filaments have also been observed close to the Golgi apparatus in mammalian cells. HIP1R functions as a linker between the actin cytoskeleton and components of the endocytic machinery, such as clathrin. So, Drubin and co-workers speculated that actin and HIP1R might be involved in vesicle-trafficking processes that occur at the TGN.

Initially, fluorescently tagged HIP1R was identified on CCVs that were closely associated with the TGN. Time-lapse microscopy then showed TGN-derived vesicles that contained both HIP1R and clathrin moving away from the TGN. These vesicles also contained the cation-dependent mannose-6-phosphate receptor (CD-MPR), which is involved in protein delivery to lysosomes.

A small interfering (si)RNA approach was then used to deplete HIP1R expression in cultured cells, which caused disruption of the TGN. Although other organelles appeared normal, the Golgi cisternae were swollen and had an accumulation of clathrin-coated buds. Furthermore, there was an increase in the size and number of lysosome-like stuctures in these cells. These observations pointed to an impairment in CCV trafficking between the TGN and lysosomes, and possibly also in lysosome function in HIP1R-depleted cells.

Actin filaments were detected associated with the TGN and CCVs in normal cells, which indicates a role for actin in CCV formation. However, HIP1R-depleted cells showed a significant increase in the number and size of these actin structures, so HIP1R might negatively regulate actin polymerization during CCV formation. Furthermore, this regulation might be required for efficient CCV release from the TGN or for subsequent movement of the CCVs. The actin-nucleating Arp2/3 complex was also identified on TGN-derived CCVs, indicating that it might stimulate, while HIP1R limits, actin assembly at these structures.

To test whether HIP1R has a role in trafficking from the TGN to lysosomes, cathepsin D — which is present in the TGN as a pro-enzyme and matures during trafficking to lysosomes — was studied using pulse-chase experiments in HIP1R-depleted cells. More of the pro-enzyme form of cathepsin D was retained in the TGN in HIP1R-depleted cells than in normal cells, an effect that was specific to the HIP1R knockdown. Treating cells with actin poisons also had a severe affect on cathepsin-D maturation, causing it to be retained in the Golgi.

The authors have therefore established a new role for actin dynamics in trafficking from the TGN to lysosomes and have shown that HIP1R is necessary for the productive coupling of actin dynamics to this pathway.