Signaling, Actin Dynamics and Endocytosis

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Endocytosis: why not wait to deubiquitinate?

Deubiquitination by the Fat facets protein - a regulator of photoreceptor differentiation during Drosophila eye development - has been found to activate endocytosis, while ubiquitination inhibits endocytosis. Surprisingly, this is the opposite effect that ubiquitination has on endocytosis of many plasma membrane proteins.     

Endocytosis and Autophagy: Exploitation or Cooperation?

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.Autophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acid-sensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ER-derived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?     

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells

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Adenovirus Endocytosis via αv Integrins Requires Phosphoinositide-3-OH Kinase

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α_v integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α_v integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α_v integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.Adenovirus entry into host cells depends on α_v integrin binding to the penton base viral coat protein (2, 20, 48). A highly mobile protrusion on the adenovirus penton base contains the arginine-glycine-aspartic acid (RGD) sequence which mediates α_v integrin binding (42). Integrins are more noted for their ability to mediate cell surface recognition of the extracellular matrix, thereby facilitating adhesion, migration (24), and cell growth and differentiation (28). These interactions have been associated with cell differentiation and tissue development, angiogenesis, wound repair, cancer, and inflammation (22).A number of cell signaling molecules that are associated with integrin-mediated cellular processes, including adhesion, survival, and motility, have recently been identified (18, 32, 34). For example, the signaling molecule pp125^FAK focal adhesion kinase (FAK) (35) is localized to clustered integrins following ligation by extracellular matrix proteins. Engagement (clustering) of integrins by its ligands increases tyrosine phosphorylation and activation of FAK (29). Potential downstream substrates of FAK are the ERK1/ERK2 mitogen-activated protein (MAP) kinases (8, 40) and phosphoinositide-3-OH kinase (PI3K) (7, 17).Recent studies have demonstrated that ligation of α_v and β1 integrins by the extracellular matrix leads to engagement of the ERK1/ERK2 MAP kinase pathway (24). Integrin-mediated regulation of the ERK1/ERK2 MAP kinase pathway results in the activation of myosin light chain kinase and subsequently to phosphorylation of myosin light chains. These molecular events culminate in enhanced cell motility. Cell motility, but not cell adhesion or spreading, can be blocked by ERK antisense oligonucleotides or by the compound PD98059, a specific inhibitor of MEK MAP kinase (24), indicating that the ERK1/ERK2 MAP kinase pathway plays a specific role in cell movement.PI3K (44) is another downstream effector of FAK. PI3K is a member of a family of lipid kinases comprised of a p85 regulatory subunit and a p110 catalytic subunit. The p85 subunit of PI3K binds directly to phosphorylated FAK (6). The products of PI3K activation, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP₃), are increased in the plasma membrane of activated but not quiescent cells and have been proposed to act as second messengers for a number of cell functions (5), including cell cycle progression (9) and cytoskeletal changes underlying the cell plasma membrane (47). PI3K activation also modulates intracellular protein trafficking (41), although a direct role of PI3K in receptor-mediated endocytosis has not been established.While integrins play an important role in adenovirus entry and in cell migration, the precise mechanisms by which these receptors promote these distinct biological functions are not known. In the studies reported here, we demonstrate that a specific signaling event is involved in the cell entry of a human viral pathogen. Evidence is provided that PI3K is activated upon adenovirus interaction with α_v integrins and that this event is required for adenovirus internalization. Surprisingly, activation of ERK1/ERK2 following integrin ligation was necessary for cell migration but not for internalization of adenovirus.     

Endocytosis: A Positive or a Negative Influence on Wnt Signalling?

Endocytosis, with subsequent targeting to lysosomes for degradation, is traditionally seen as a way for cells to terminate signalling. However, in a few instances, endocytosis has been demonstrated to contribute positively to signalling. Here we review recent work on the role of endocytosis in Wnt signalling. Biochemical evidence suggests that the branch of Wnt signalling that controls planar cell polarity (PCP) does require endocytosis, although how endocytosis of Frizzled receptors is translated into PCP in vivo remains unknown. With respect to the main signalling branch (called the canonical or beta-catenin pathway), the literature is divided as to whether endocytosis is required. Results of in vivo experiments are inconclusive because of the toxic side-effects of blocking endocytosis. Some results with cultured cells suggest the need for endocytosis in canonical signalling; however, it remains unclear whether the ligand-receptor complex must enter the cell by clathrin-mediated or caveolae-mediated endocytosis in order to signal. Means of specifically altering Wnt trafficking as well as of tracking the internalization route in different cell types are needed.     

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.     

Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with μ2

Background

Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors.

Methodology/Principal Findings

Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product.

Conclusions/Significance

These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.     

丙酸睾酮对鸡的法氏囊表面上皮内吞作用(Endocytosis)的影响

在莱杭雏鸡和仔鸡的肛唇上进行胶质碳处理,证明法氏囊附着滤泡上皮具有内吞作用。被内吞的碳粒24小时以后,主要存在于淋巴滤泡髓部的星状细胞及胞间隙中。在法氏囊发育的不同时期,用丙酸睾酮处理,均可损害附着滤泡上皮细胞。它们的亚微结构发生了变化,同时正常的内吞作用也受到明显地抑制。特别是用高剂量的丙酸睾酮处理以后,附着滤泡上皮细胞的内吞作用受到完全抑制。此外也揭示了丙酸睾酮处理以后,法氏囊淋巴滤泡表面的形态变化与法氏囊自然退化的情形大致相似。进一步证明法氏囊的自然退化与体内性激素浓度升高有关。     

Endocytosis mediated by monocyte and macrophage membrane lectins — application to antiviral drug targeting

Sugar receptors, or membrane lectins, have been evidenced at the surface of various normal and tumour cells using fluoresceinylated neoglycoproteins (glycosylated bovine serum albumin (BSA)). By flow cytometry we have shown that macrophages bind and internalize mannosylated and 6-phosphomannosylated ligands in acidic compartments. Freshly isolated monocytes and U937, a promonocytic cell line, lack a mannose-specific receptor, but express mannose-6-phosphate (Man-6P) membrane lectin.Neoglycoproteins are potent drug carriers: muramyl dipeptide (MDP), an immunoactivator, when bound to Man-BSA or Man-6P-BSA, is 100 times more efficient than free MDP in activating macrophages; in vivo, it enables eradication of lung metastases in mice. Recently, neutral glycosylated biodegradable and non-immunogenic polymers, were synthesized and found to be as efficient as neoglycoproteins. Antiviral drug conjugates were more active than the free drug, inhibiting the multiplication of virus (herpes) in human macrophages in vitro.     

Activity-Dependent Bulk Synaptic Vesicle Endocytosis—A Fast,High Capacity Membrane Retrieval Mechanism

Central nerve terminals are placed under considerable stress during intense stimulation due to large numbers of synaptic vesicles (SVs) fusing with the plasma membrane. Classical clathrin-dependent SV endocytosis cannot correct for the large increase in nerve terminal surface area in the short term, due to its slow kinetics and low capacity. During such intense stimulation, an additional SV retrieval pathway is recruited called bulk endocytosis. Recent studies have shown that bulk endocytosis fulfils all of the physiological requirements to remedy the acute changes in nerve terminal surface area to allow the nerve terminal to continue to function. This review will summarise the recent developments in the field that characterise the physiology of bulk endocytosis which show that it is a fast, activity-dependent and high capacity mechanism that is essential for the function of central nerve terminals.     

TGF-β Signaling Is Associated with Endocytosis at the Pocket Region of the Primary Cilium

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Tyrosine-based Signal Mediates LRP6 Receptor Endocytosis and Desensitization of Wnt/β-Catenin Pathway Signaling

Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.     

Endocytosis via caveolae: alternative pathway with distinct cellular compartments to avoid lysosomal degradation?

Endocytosis – the uptake of extracellular ligands, soluble molecules, protein and lipids from the extracellular surface – is a vital process, comprising multiple mechanisms, including phagocytosis, macropinocytosis, clathrin-dependent and clathrin-independent uptake such as caveolae-mediated and non-caveolar raft-dependent endocytosis. The best-studied endocytotic pathway for internalizing both bulk membrane and specific proteins is the clathrin-mediated endocytosis. Although many papers were published about the caveolar endocytosis, it is still not known whether it represents an alternative pathway with distinct cellular compartments to avoid lysosomal degradation or ligands taken up by caveolae can also be targeted to late endosomes/lysosomes. In this paper, we summarize data available about caveolar endocytosis. We are especially focussing on the intracellular route of caveolae and providing data supporting that caveolar endocytosis can join to the classical endocytotic pathway.