Proteins and mechanisms involved in endosomal sorting of the epidermal growth factor receptor

Signaling from growth factor receptors is tightly regulated. Failure of this regulation can lead to overstimulation of growth, proliferation and differentiation, and is often observed in cancer. The epidermal growth factor receptor (EGFR) is desensitized by internalization and degradation in lysosomes. Internalization takes place rapidly upon ligand induced activation, whereas degradation depends on retained ligand binding and subsequent sorting to inner vesicles on multivesicular bodies (MVBs). Using electron-microscopy, confocal immunofluorescence microscopy and standard molecular biology techniques, we have investigated endocytosis and endosomal sorting of EGFR. Endocytosis of the activated EGFR is believed to depend on ubiquitination of the receptor. Ubiquitination depends on the ubiquitin ligase Cbl, and we show that the direct interaction between EGFR and Cbl at the phosphorylated tyrosine residue 1045 (pY1045) of the EGFR is sufficient for endocytosis of the EGFR, but not sufficient for sorting to MVBs and degradation. On endosomes, different membrane domains are involved in sorting of the endocytosed EGFR, and we found that two different electron dense coats are involved, one coat containing clathrin and Hrs, and another coat devoid of clathrin and Hrs. Hrs is involved in sorting of ubiquitinated receptors for degradation, and we further found that the ubiquitination induced by direct interaction between EGFR and Cbl at the pY1045 of the EGFR is necessary for co-localization between the EGFR and Hrs. Finally, we found the non-receptor tyrosine kinase Ack1, which is often over-expressed in cancer, to be involved in EGFR endocytosis, most likely at the level of endosomal sorting. Ack1 colocalized with the EGFR on early endosomes and overexpression of Ack1 retained the EGFR on the limiting membrane of early endosomes and inhibited sorting of the EGFR to inner vesicles of MVBs

Ubiquitination in Control of EGF Receptor Endocytosis

Growth factors and growth factor receptors comprise a signaling network regulating cell differentiation and growth. Tightly regulation of this network is important to avoid excessive signaling and cell growth, which may result in cancer. It is therefore important to gain insight into how this network is regulated and into mechanisms of signal termination. Signaling from epidermal growth factor receptor (EGFR) is terminated by downregulating the receptor from the plasma membrane by clathrin-mediated endocytosis and subsequent lysosomal degradation. The mechanisms involved in EGFR endocytosis are controversial. In this work we have investigated which adaptor proteins are important for EGFR endocytosis and whether the posttranslational modification termed ubiquitination is involved in EGFR endocytosis. We have shown that EGFR is concentrated in clathrin coated pits at the plasma membrane upon activation with EGF, and that the adaptor proteins Grb2 and Cbl are important for this process. Cbl mediates ubiquitination of activated EGFR, and we found that by inhibiting the activity of Cbl in different ways, EGFR was less effectively ubiquitinated and also less effectively concentrated in clathrin coated pits. We also studied whether the ubiquitin binding protein Epsin 1 could function as an adaptor for recruiting ubiquitinated EGFR into clathrin coated pits. We found that depleting cells of Epsin 1 by RNA interference reduced the recruitment of EGFR to clathrin coated pits, and consistently, EGFR endocytosis was inhibited. Using recombinant DNA-technology we covalently fused ubiquitin to EGFR to study whether ubiquitin could drive endocytosis in the absence of EGFR activation. This study indeed demonstrated that ubiquitin per se is sufficient to mediate endocytosis of EGFR from clathrin coated pits. In conclusion, this work presents evidence supporting ubiquitination as a mechanism controlling clathrin-mediated endocytosis of EGFR

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization

The role of clathrin in post-golgi trafficking in toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle

Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit

Dynamin, the GTPase required for clathrin-mediated endocytosis, is recruited to clathrin-coated pits in two sequential phases. The first is associated with coated pit maturation; the second, with fission of the membrane neck of a coated pit. Using gene-edited cells that express dynamin2-EGFP instead of dynamin2 and live-cell TIRF imaging with single-molecule EGFP sensitivity and high temporal resolution, we detected the arrival of dynamin at coated pits and defined dynamin dimers as the preferred assembly unit. We also used live-cell spinning-disk confocal microscopy calibrated by single-molecule EGFP detection to determine the number of dynamins recruited to the coated pits. A large fraction of budding coated pits recruit between 26 and 40 dynamins (between 1 and 1.5 helical turns of a dynamin collar) during the recruitment phase associated with neck fission; 26 are enough for coated vesicle release in cells partially depleted of dynamin by RNA interference. We discuss how these results restrict models for the mechanism of dynamin-mediated membrane scission

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.Moreno-Layseca et al. identify Swip1 as an integrin-specific endocytic adaptor controlling the dynamics of integrin adhesion complexes as well as the migration and invasion of breast cancer cells

Cargo-specific recruitment in clathrin and dynamin-independent endocytosis

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis, and cancer invasion and is often hijacked by viral infections. Unlike clathrin-mediated endocytosis, which exploits cargo-specific adaptors for selective protein internalization, the clathrin and dynamin-independent endocytic pathway (CLIC-GEEC, CG-pathway) has until now been considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. Although the core molecular players of CG endocytosis have been recently defined, no cargo-specific adaptors are known and evidence of selective protein uptake into the pathway is lacking. Here, we identify the first cargo-specific adaptor for CG-endocytosis and demonstrate its clinical relevance in breast cancer progression. By combining unbiased molecular characterization and super-resolution imaging, we identified the actin-binding protein swiprosin-1 (EFHD2) as a cargo-specific adaptor regulating integrin internalization via the CG-pathway. Swiprosin-1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery, IRSp53 and actin. Swiprosin-1 is critical for integrin endocytosis, but not for other CG-cargo and supports integrin-dependent cancer cell migration and invasion, with clinically relevant implications for breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG-pathway and opens the possibility to discover more adaptors regulating it

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al