A direct role for Met endocytosis in tumorigenesis

Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants' anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.     

A critical role for endocytosis in Wnt signaling

Background  

The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation.     

Clathrin-independent endocytosis: A cargo-centric view

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.     

AtEHDs in endocytosis

Endocytosis regulates many important and diverse processes in eukaryotic life. EH domain containing proteins function as regulators of endocytosis through protein-protein interactions. Several interactors of mammalian EHDs were identified, including clathrin machinery components. The four human EHD proteins share high homology at the protein level and possess similar domains, but appear to be involved in different stages of intracellular trafficking. EHD1 regulates recycling through the endocytic recycling compartment (ERC). EHD2 has been found to inhibit internalization in mammalians when overexpressed.We have recently investigated the importance of EH domain containing proteins in plant endocytosis. We were able to show that both of the Arabidopsis EHD homologs, termed AtEHD1 and AtEHD2, play important roles in plant endocytosis. Knockdown of AtEHD1 delayed internalization, and overexpression of AtEHD2 inhibited endocytosis. Thus, the function of plant EHDs is highly homologous to that of mammalian EHDs.Key words: endocytosis, endosome, EH domain, EHD1, EHD2, recycling     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Lipid-mediated endocytosis

Receptor-mediated endocytosis is used by a number of viruses and toxins to gain entry into cells. Some have evolved to use specific lipids in the plasma membrane as their receptors. They include bacterial toxins such as Shiga and Cholera toxin and viruses such as mouse polyoma virus and simian virus 40. Through multivalent binding to glycosphingolipids, they induce lipid clustering and changes in membrane properties. Internalization occurs by unusual endocytic mechanisms involving lipid rafts, induction of membrane curvature, trans-bilayer coupling, and activation of signaling pathways. Once delivered to early endosomes, they follow diverse intracellular routes to the lumen of the ER, from which they penetrate into the cytosol. The role of the lipid receptors is central in these well-studied processes.     

Yeast endocytosis

The presence of an endocytic pathway in cells from a wide range of species and the conservation of the proteins involved in this process throughout evolution suggest that endocytosis is of fundamental importance for the eukaryotic cell. However, some surprising recent results have shown that both Dictyostelium discoideum and Saccharomyces cerevisiae can live under laboratory conditions with substantially reduced levels of endocytosis. In this review, I concentrate on endocytosis in S. cerevisiae. Recent progress in the study of intermediates of the endocytic pathway and of mutants affecting the endocytic pathway make this organism an interesting model with which to study the mechanism and functions of endocytosis.     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Ephective endocytosis

Two important new reports identify endocytosis of EphB-ephrinB complexes as a mechanism for switching between cell-cell adhesion and repulsion following plasma membrane contact. Together with the previously described shedding of ephrinA following EphA engagement, these findings resolve the paradox of how an adhesive receptor-ligand interaction generates a repulsive cellular response.     

Structural perturbations in the plasma membrane during concanavalin A endocytosis

Endocytosis of Concanavalin A, triggered by its interaction with the surface of cells from a murine plasmocytoma line, is characterised by various steps which can be visualised by cytochemistry and freeze-fracturing. Plasma membrane internalisation is initiated by clustering of Concanavalin A receptors and by formation of intramembraneous particle necklaces, which were observed on fracture faces. Subsequent perturbation of the lipid bilayer precedes the fusion and formation of closed vesicles.     

A potent endocytosis inhibitor Ikarugamycin up-regulates TNF production

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.     

A functional GFP fusion for imaging clathrin-mediated endocytosis

The ability to localize proteins of interest in live cells through imaging inherently fluorescent protein tags has provided an unprecedented level of information on cellular organization. However, there are numerous cases where fluorescent tags alter the localization and/or function of the proteins to which they are appended. Clathrin-mediated endocytosis from the plasma membrane is a physiologically important process evolutionarily conserved from yeast to humans. Some proteins that are associated with the machinery of clathrin-mediated endocytosis have been tagged with fluorescent proteins. However, it has not yet been possible to study this process through a protein marker that is specific to this step and still fully functional when linked to a fluorescent protein. In this study, we present the first demonstration that one of these proteins, in this case a green fluorescent protein (GFP) fusion to α-adaptin, a marker of the adaptor protein-2 complex, functionally complements knockdown of endogenous protein through small interfering RNA silencing. GFP–α-adaptin, as well as the techniques used to test the fusion protein, represents an important contribution to the cell biologist's toolbox, which will permit a greater understanding of vesicle trafficking in live cells.     

SCAMP1 function in endocytosis

Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.     

Clathrin-mediated endocytosis in budding yeast

Clathrin-mediated endocytosis in the budding yeast Saccharomyces cerevisiae involves the ordered recruitment, activity and disassembly of nearly 60 proteins at distinct sites on the plasma membrane. Two-color live-cell fluorescence microscopy has proven to be invaluable for in vivo analysis of endocytic proteins: identifying new components, determining the order of protein arrival and dissociation, and revealing even very subtle mutant phenotypes. Yeast genetics and functional genomics facilitate identification of complex interaction networks between endocytic proteins and their regulators. Quantitative datasets produced by these various analyses have made theoretical modeling possible. Here, we discuss recent findings on budding yeast endocytosis that have advanced our knowledge of how -60 endocytic proteins are recruited, perform their functions, are regulated by lipid and protein modifications, and are disassembled, all with remarkable regularity.