Regulating Endocytic Trafficking from the Late Endosome Essay
Regulating Endocytic Trafficking from the Late Endosome
EssaRab7 has been detected in many tissues by immunofluorescence method. With the use of immunofluorescence and confocal microscopy, the intracellular distribution of Rab7 can be examined. At the molecular level, Rab7 has been known to control endosomal trafficking via its GTPase activity. It has been suggested that active GTP-bound Rab7 increases the velocity of dynein-mediated endosomal transport along the microtubules. Several diverse sets of physiological mechanisms of the cell have been demonstrated to be Rab7 mediated. The glucose load in the type 2 diabetics can thus be handled in the manner indicated, and hence this could be a possible signal mediated trafficking mechanism that may be involved in the control of extracellular glucose levels in these individuals. Regulating Endocytic Trafficking from the Late Endosome Essay. In this experiment, localization of Rab7 protein vesicles through a novel method has been demonstrated, and this could serve as a novel method of the intervention of the pathway of cellular expressions, so a drug target can be developed to treat these individuals more effectively.
The beta-cells in the pancreas secrete primary polypeptide hormone product that is packaged into large membrane-bound secretory vesicles or beta-granules at the trans-face of the Golgi complex for release via the regulated secretory pathway (Feng and Arvan, 2003, 31486-31494). Most beta-granules reside in an internal storage pool, but in response to a physiological stimulus such as elevated blood glucose, a sub-population of these granules is transported to the plasma membrane and undergo exocytosis, resulting in the extracellular secretion of insulin (Kalina et al., 1989, 19-23). The Rab family of small GTP binding proteins represents key components of vesicular trafficking, mediating a range of diverse functions, such as vesicle formation, vesicle transport, organelle motility, vesicle docking and fusion, and exocytosis and endocytosis.y
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Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), which phosphorylates phosphatidylinositol-3-phosphate (PI3P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells.
Methods
Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8), bile acids, and cigarette toxin was determined. Regulating Endocytic Trafficking from the Late Endosome Essay.
Results
PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini.
Conclusions
These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens, leading to the pathogenicity of acute pancreatitis.
Abbreviations used in this paper
Summary
This brief overview of endocytic trafficking is written in honor of Renate Fuchs, who retires this year. In the mid-80s, Renate pioneered studies on the ion-conducting properties of the recently discovered early and late endosomes and the mechanisms governing endosomal acidification. As described in this review, after uptake through one of many mechanistically distinct endocytic pathways, internalized proteins merge into a common early/sorting endosome. From there they again diverge along distinct sorting pathways, back to the cell surface, on to the trans-Golgi network or across polarized cells.Regulating Endocytic Trafficking from the Late Endosome Essay. Other transmembrane receptors are packaged into intraluminal vesicles of late endosomes/multivesicular bodies that eventually fuse with and deliver their content to lysosomes for degradation. Endosomal acidification, in part, determines sorting along this pathway. We describe other sorting machinery and mechanisms, as well as the rab proteins and phosphatidylinositol lipids that serve to dynamically define membrane compartments along the endocytic pathway.
Introduction
Endocytosis, the process by which cells internalize macromolecules and surface proteins, was first discovered with the advancement of electron microscopy that enabled visualization of the specialized membrane domains responsible for two mechanistically and morphologically distinct pathways: clathrin-mediated endocytosis (CME) [1] and caveolae uptake [2] (see Table 1 for a glossary of terms and abbreviations used in this review). Selective inhibition of these two pathways later led to the discovery of cholesterol-sensitive clathrin- and caveolae-independent pathways [3–5], and more recently, the large capacity pathway involving clathrin independent carriers (CLIC) and glycophosphatidylinositol-anchored protein-enriched endosomal compartments (GEEC), termed the CLIC/GEEC pathway [6]. Regulating Endocytic Trafficking from the Late Endosome Essay.
Table 1
Glossary of terms and abbreviations
| Abbreviation | Term/Definition | Role/Function |
|---|---|---|
| AP2 | Adaptor Protein 2 | Recruits clathrin and cargo to growing clathrin coated pits |
| Arf6-dependent pathway | ADP-ribosylation factor 6 -dependent pathway | A clathrin-independent endocytic pathway |
| BAR domain-containing proteins | Bin/Amphiphysin/Rvs domain-containing proteins | proteins that can sense and generate membrane curvature |
| CavME | Caveolae-Mediated Endocytosis | An endocytic pathway |
| CCP/CCV | Clathrin-Coated Pit or Vesicle | The vehicles of clathrin-mediated endocytosis |
| CLASP | Clathrin Associated Sorting Proteins | Cargo specific adaptor proteins that work together with AP2 complexes |
| CIE | Clathrin-Independent Endocytosis | Describes alternate routes of endocytosis |
| CLIC/GEEC pathway | CLathrin-Independent Carriers, GPI-AP-Enriched Endocytic Compartments | A clathrin-independent endocytic pathway |
| CME | Clathrin-Mediated Endocytosis | The major pathway for endocytic uptake |
| EEA1 | Early Endosome Antigen 1 | An early endosome associated scaffold protein |
| ESCRT | Endosomal Sorting Complexes Required for Transport | 4 complexes mediate formation in intraluminal vesicles during endosome maturation |
| FVYE domain | Fab-1, YGL023, Vps27, and EEA1 domain | Protein domain that interacts with PI(3)P a phosphatidyl inositol lipid |
| GAP | GTPase-Activating Protein | Inactivates small GTPases by catalyzing GTP hydrolysis |
| GEF | Guanine nucleotide Exchange Factor | Activated small GTPases by catalyzing GTP/GDP binding |
| GPI-AP | Glycosylphosphatidylinositol-anchored Proteins | endocytic cargo |
| HSC70 | Heat Shock Cognate 70 | Also functions as the uncoating ATPase, to disassemble clathrin |
| ILV | Intraluminal Vesicle | Substructure of early/late endosomes. Sorts transmembrane cargo for degradation in lysosomes |
| MVB | Multivesicular body | A late endosome with cargo sorted into intraluminal vesicles for delivery to lysosomes |
| PH domain | Pleckstrin Homology domain | Protein domains that mediate specific PIP interactions |
| PIP | Phosphatidylinositol phospholipid | A phospholipid species readily interconvertable by phosphorylation/dephosphorylatio of the inositol head group |
| PI(4,5)P2 | Phosphatidylinositol-4,5-bisPhosphate | Phospholipid component enriched in the plasma membrane |
| PI(3) | Phosphatidylinositol-3-Phosphate | Phospholipid component enriched in early endosome membranes |
| PX domain | PhoX homology domain | Protein domain required for specific PIP interactions |
| SH3 domain | SRC Homology 3 domain | Protein domains required for protein-protein interaction |
| SNX | Sorting Nexin family Proteins | Scaffolding and curvature generating proteins involved in endocytic trafficking |
| TGN | Trans-Golgi Network | A post-Golgi sorting compartment |
After molecules have been internalized through one of these different endocytic pathways they traffic through and are sorted by a pleiomorphic series of tubulovesicular compartments, collectively called endosomes [7]. Internalized macromolecules and surface proteins can have many different fates: They can be recycled back to the plasma membrane, delivered to the lysosomes for degradation, or in polarized cells sent across the cell through a process called transcytosis, which is important for transport across epithelia, endothelia and the blood brain barrier [8]. Endosomal compartments undergo maturation from early to late endosomes, which involves decreasing luminal pH, altering key phosphatidylinositol lipids through regulation by lipid kinases and phosphatases, and differential recruitment and activation of Rab-family GTPases.
Following the initial discovery of these pathways and their trafficking, it was then determined that each endocytic pathway fulfills multiple critical cellular functions. Cells communicate with each other and their environment through endocytosis. Consequently, endocytosis regulates the levels of many essential surface proteins and transporters in human health and disease, such as the glucose transporters that maintain serum glucose levels, proton pumps that control stomach acidification, or sodium channels that control cell homeostasis [9]. Furthermore, endocytosis regulates signaling from surface receptors like G-protein coupled receptors [10] and receptor tyrosine kinases [11]. Finally, endocytosis regulates cell-cell and cell-matrix interactions through uptake of integrins and adhesion molecules [12]. Regulating Endocytic Trafficking from the Late Endosome Essay. Since the discovery of endocytosis several decades ago, its complex and critical role in human physiology and pathology has become increasingly appreciated and better understood. This review briefly summarizes our current state of knowledge about cargo sorting and trafficking along the endocytic pathway.
Multiple Mechanisms for uptake into cells
Clathrin-mediated endocytosis
The most studied and hence, well-characterized endocytic mechanism is clathrin-mediated endocytosis (Figure 1), which occurs through clathrin-coated pits (CCPs) and clathrin coated vesicles (CCVs), first observed >50 years ago by thin section electron microscopy [1]. CME was first found to play an important role in low-density lipoprotein [13] and transferrin uptake [14] upon binding to their respective receptors (hereafter referred to as ‘cargo’). The principle components of the CCVs are the heavy and light chains of clathrin [15], from which the pathway acquires its name, and the four subunits of the heterotetrameric adaptor protein 2 (AP2) complex [16]. The AP2 complex links the clathrin coat to the membrane bilayer and is also the principle cargo-recognition molecule [17]. There are other specialized adaptor proteins, collectively called “CLASPs” (for clathrin associated sorting proteins) [18, 19], which each recognize distinct sorting motifs on their respective cargo receptors. These cargo-specific adaptor proteins often interact with both clathrin and the AP2 complex, increasing the repertoire of cargo that can be sorted.
Schematic representation of the different endocytic pathways described in mammalian cells. The pathways can be broken into two main groups, dynamin-dependent and clathrin- and dynamin-independent pathways. Ligands known to traffic through each pathway, some of which have disease implications, are labeled along with the important molecular players of each pathway. The representation was inspired by [93].
Clathrin-mediated endocytosis proceeds through multiple stages: CCP initiation, cargo-selection, CCP growth and maturation, scission and CCV release, and, finally, uncoating. CCP assembly is initiated by AP2 complexes that are recruited to the plasma membrane-enriched phosphatidylinositol lipid, PI(4,5)P2 [19]. AP2 complexes then rapidly recruit clathrin [20]. Other scaffolding molecules (e.g. FCHo, eps15, and/or intersectins) also assemble early and may play a role in either nucleating and/or stabilizing nascent CCPs [20, 21]. Regulating Endocytic Trafficking from the Late Endosome Essay. Although clathrin has been shown to spontaneously assemble into closed cages in vitro [22], in cells, other curvature generating proteins must be recruited to nascent CCP’s for efficient budding. For example, the intrinsically curved, BAR (Bin-Amphiphysin-Rvs) domain-containing proteins can create increasingly deeper curvature and are thought to be required for progression of the clathrin-coated pit [23]. As the nascent pits grow, AP2 and other cargo-specific adaptor proteins recruit and concentrate cargo. Clathrin polymerization stabilizes the curvature of the pit; however other factors recruited to AP2 complexes are also required for efficient curvature generation and their subsequent invagination [24].
Release of mature clathrin-coated vesicles from the plasma membrane depends on the large GTPase dynamin [25]. Dynamin is recruited to clathrin-coated pits by BAR domain-containing proteins [26] such as amphiphysin, endophilin, and sorting nexin 9, which also encode SRC homology 3 (SH3) domains that bind to dynamin’s proline-rich domain. Dynamin assembles into collar-like structures encircling the necks of deeply invaginated pits and undergoes GTP hydrolysis to drive membrane fission [25]. Finally, once the vesicle is detached from the plasma membrane the clathrin coat is disassembled by the ATPase, heat shock cognate 70 (HSC70) and its cofactor auxilin [27, 28]. This allows the now uncoated vesicle to travel and fuse with its target endosome.
Caveolae-mediated endocytosis
Caveolae-mediated endocytosis (CavME), which was also first discovered ~60 years ago by thin section electron microscopy [2], is the second most well-characterized and studied endocytic pathway (Figure 1). CavME has been found to be important in transcytotic trafficking across endothelia as well as mechanosensing and lipid regulation [29]. Caveolae, the site of CavME, are flask or omega-shaped plasma membrane invaginations with a diameter of 50–100 nm and are abundantly present on many but not all eukaryotic plasma membranes [30]. Biochemical studies have revealed that caveolae are detergent resistant, highly hydrophobic membrane domains enriched in cholesterol and sphingolipids [31, 32]. In addition to their role in endocytosis, caveolae have been implicated as signaling platforms, regulators of lipid metabolism and in cell surface tension sensing [33].
The main structural proteins of caveolae are members of the caveolin protein family, the most common being caveolin-1. Caveolin-1 is a small integral membrane protein that is inserted into the inner leaflet of the membrane bilayer. The cytosolic N-terminal region of caveolin-1 binds to cholesterol and functions as a scaffolding domain that binds to important signaling molecules [33]. Once thought to be sufficient for the formation of caveolae, it is now known that caveolins co-assemble with cytosolic coat proteins, called cavins, to form these structures [34].
Live cell microscopy studies have revealed that caveolae are static structures and that CavMe is highly regulated and triggered by ligand binding to cargo receptors concentrated in caveolae [29]. The steps involved in CavME are not as well understood as those involved in CME. However, budding of caveolae from the plasma membrane is known to be regulated by kinases and phosphatases [35], as numerous studies have shown that their chemical inhibition either inhibits (in the case of kinase inhibitors) or enhances (in the case of phosphatase inhibitors) CavME [36, 37]. Finally, like CME, dynamin is required to pinch off caveolae vesicles from the plasma membrane [38].
Clathrin-independent endocytosis
More recently, it has become clear that other mechanistically distinct endocytic pathways mediate uptake of different subsets of signaling, adhesion and nutrient receptors, as well as regulate the surface expression of membrane transporters. These pathways have been shown to be clathrin-independent endocytic (CIE) pathways, and as the name infers, the endocytic vesicles/tubules involved in CIE have no distinct coat and are not easily detected by EM. Thus, these pathways were first discovered by virtue of their resistance to inhibitors that block CME and CavME [3, 4, 39].
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The term CIE encompasses several pathways (Figure 1, reviewed in [40–42]): i) An endophilin-, dynamin- and RhoA-dependent pathway that was first identified for its role in interleukin-2 receptor endocytosis [5] and recently shown to mediate uptake of many other cytokine receptors and their constituents [43]. ii) A recently discovered clathrin- and dynamin-independent pathway that involves the small GTPases Rac1 and Cdc42 and leads to the actin-dependent formation of so-called clathrin-independent carriers (CLICs) [44], which fuse to form a specialized early endosomal compartment called the GPI-AP enriched endosomal compartments (GEECs) [6, 42]. Hence, this process is termed the CLIC/GEEC pathway. iii) A pathway depending on the small GTPase, Arf6, that was first shown to mediate uptake and recycling of the Major Histocompatibility Antigen I [41]. Regulating Endocytic Trafficking from the Late Endosome Essay. Arf6 activates phosphatidylinositol-4-phosphate 5-kinase to produce PI(4,5)P2 to stimulate actin assembly and drive endocytosis [45]. And, iv) a pathway dependent on curvature-generating, membrane-anchored proteins, called flotillins [46, 47]. The degree of overlap of these pathways is still incompletely understood and, somewhat disputed [48]. Certainly, they carry overlapping cargo molecules, for example the GPI-anchored protein, CD59, is taken up by both the CLIC/GEEC and in a flottilin-dependent manner in HeLa cells. Similarly, the transmembrane protein, CD44 is a cargo of both the Arf6-pathway and the CLIC/GEEC pathway [49]. Thus, it remains unclear as to whether they represent mechanistically distinct pathways or cell type and/or experimentally induced variations of the same pathway. Additional molecular machinery and mechanistic insights are needed to resolve these issues and better define these pathways.
The role of these CIE pathways in the cell and the extent to which they contribute to the endocytic capacity of the cell remains unclear. Thus, whereas some studies suggest they are the major pathways for bulk uptake [6], other authors have suggested that CME can account for virtually all bulk uptake [50], inferring that CIE might be induced only upon disruption of CME. However, a recent survey of endocytic activities in 29 different non-small cell lung cancer cells revealed that CIE pathways are differentially regulated relative to CME and CavME, providing strong evidence for their autonomy and functional importance [51].
Endosomal Trafficking
Once internalized into cells through any of the multiple endocytic pathways, cargoes (receptors and their bound ligands) then merge into the common endosomal network. The endosomal network is a dynamic and interconnected “highway” system that allows for the vectorial trafficking and transfer of cargoes between distinct membrane-bound compartments. The function of the endosomal network is to collect internalized cargoes, sort, and disseminate them to their final destinations [44]. Upon entering the cell, primary endocytic vesicles undergo multiple rounds of homotypic fusion [52] to form early/sorting endosomes. Within early endosomes initial sorting decisions are made and the fates of the internalized receptors are decided. Ultimately, cargoes can be recycled back to the plasma membrane, sent to the trans-Golgi network (TGN) via retrograde traffic, or sorted to the lysosome for degradation (Figure 2). The regulation of membrane traffic and cargo sorting is achieved through the tight spatial and temporal control of endosomal identity. Regulating Endocytic Trafficking from the Late Endosome Essay.
