Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.

Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.

Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2.

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell-cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.

Rab and Arf G proteins in endosomal trafficking and cell surface homeostasis.

The dynamics of membrane fusion, fission, cargo sorting and organelle maturation in endosomal membrane systems is regulated by Rab and Arf small G proteins. Defining the regulators, effectors and sites of action for each Rab and Arf will enhance our understanding of how endocytic membrane traffic is orchestrated and functions in differentiated cell types. Recent work has also shown how Rab35 and Arf6 might serve as input sensors for 2 forms of endocytosis to balance membrane trafficking to preserve cell surface homeostasis.

Rab and Arf G proteins in endosomal trafficking.

Endocytosis is a fundamental process that cells use to remove receptors, extracellular material, plasma membrane proteins and lipids from the cell surface. After entry into cells, the cargo proteins are subsequently trafficked to late endosomes and lysosomes for degradation, to the Golgi complex, or to recycling endosomes for return to the plasma membrane. Small G proteins in the Rab and Arf family are present on endosomes and coordinate the trafficking of cargo proteins. Here we describe some basic experimental approaches to begin to study the endosomal trafficking of a given cell surface protein.

Search for inhibitors of endocytosis: Intended specificity and unintended consequences.

We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to "classify" entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.

Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis.

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.