Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (alpha- and beta 2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.     

Capturing the mechanics of clathrin-mediated endocytosis

Clathrin-mediated endocytosis enables selective uptake of molecules into cells in response to changing cellular needs. It occurs through assembly of coat components around the plasma membrane that determine vesicle contents and facilitate membrane bending to form a clathrin-coated transport vesicle. In this review we discuss recent cryo-electron microscopy structures that have captured a series of events in the life cycle of a clathrin-coated vesicle. Both single particle analysis and tomography approaches have revealed details of the clathrin lattice structure itself, how AP2 may interface with clathrin within a coated vesicle and the importance of PIP2 binding for assembly of the yeast adaptors Sla2 and Ent1 on the membrane. Within cells, cryo-electron tomography of clathrin in flat lattices and high-speed AFM studies provided new insights into how clathrin morphology can adapt during CCV formation. Thus, key mechanical processes driving clathrin-mediated endocytosis have been captured through multiple techniques working in partnership.     

ATG16L1 meets ATG9 in recycling endosomes

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Commentary: ATG16L1 meets ATG9 in recycling endosomes: Additional roles for the plasma membrane and endocytosis in autophagosome biogenesis

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis

Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.     

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis

The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.     

Clathrin exchange during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Adp-Ribosylation Factor 6 and Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells

We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6–Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6–T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6–Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6–Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.     

Auxilin depletion causes self-assembly of clathrin into membraneless cages in vivo

Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an ∼50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an ∼1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.     

Clathrin-mediated endocytosis: the gateway into plant cells

Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, hormonal signaling and communication with the environment including nutrient delivery, toxin avoidance, and pathogen defense. The major endocytic mechanism in plants depends on the coat protein clathrin. It starts by clathrin-coated vesicle formation at the plasma membrane, where specific cargoes are recognized and packaged for internalization. Recently, genetic, biochemical and advanced microscopy studies provided initial insights into mechanisms and roles of clathrin-mediated endocytosis in plants. Here we summarize the present state of knowledge and compare mechanisms of clathrin-mediated endocytosis in plants with animal and yeast paradigms as well as review plant-specific regulations and roles of this process.     

Autophagosome precursor maturation requires homotypic fusion

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.     

Endocytosis of beta1 integrins is an early event in migration promoted by the cell adhesion molecule L1

Directional cell motility is a complex process requiring orchestration of signals from diverse cell adhesion receptors for proper organization of neuronal groups in the brain. The L1 cell adhesion molecule potentiates integrin-dependent migration of neuronal cells and stimulates integrin endocytosis but its mechanism of action is unclear. The hypothesis was investigated that L1 stimulates cell motility by modulating surface levels of integrins through intracellular trafficking using a model cell system. Antibody-induced clustering of L1, which mimics ligand binding, induced formation of cell surface complexes of L1 and beta1 integrins in L1-expressing HEK293 cells. L1 formed cell surface complexes with integrin beta1 and alpha3 subunits but not with integrin alpha1. Following cell surface clustering, beta1 integrins and L1 became rapidly internalized into Rab5+ early endosomes. Internalization of L1 and beta1 integrins was prevented by treatment with monodansyl cadaverine (MDC), an inhibitor of clathrin-dependent endocytosis, and by deletion of the AP2/clathrin binding motif (RSLE) from the L1 cytoplasmic domain. MDC treatment coordinately inhibited L1-potentiated haptotactic migration of HEK293 cells to fibronectin in Transwell assays. These results suggested that downregulation of adhesive complexes of L1 and beta1 integrin at the plasma membrane by clathrin-mediated endocytosis is a potential mechanism for enhancing cell motility.     

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.