Unconventional Functions for Clathrin,ESCRTs, and Other Endocytic Regulators in the Cytoskeleton,Cell Cycle,Nucleus, and Beyond: Links to Human Disease

The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease.The discovery and characterization of clathrin (Pearse 1975) initiated molecular definition of the many endocytosis regulators described in this collection, which mediate the clathrin-dependent and -independent pathways for membrane internalization (see Kirchhausen et al. 2014; Mayor et al. 2014; Merrifield and Kaksonen 2014). In accompanying reviews, we have seen how these endocytic pathways influence nutrition and metabolism (see Antonescu et al. 2014), signal transduction (see Bökel and Brand 2014; Di Fiore and von Zastrow 2014), neuronal function (see Morgan et al. 2013; Cosker and Segal 2014), infection and immunity (see ten Broeke et al. 2013; Cossart and Helenius 2014), tissue polarity and development (see Eaton and Martin-Belmonte 2014; Gonzalez-Gaitan and Jülicher 2014), and migration and metastasis (see Mellman and Yarden 2013). Recently, it has been established that some endocytic regulators have molecular properties that expand their functions beyond endocytosis. These include molecular interactions that affect the microtubule and actin cytoskeletons, nuclear translocation that influences gene regulation, and the formation of membrane-associated scaffolds that serve as signaling and sorting platforms. Through these diverse nonendocytic functions, endocytosis regulators play additional roles in cell division, pathogen infection, cell adhesion, and oncogenesis. In this article, we review the nonconventional behavior of endocytic regulators, first discussing the molecular properties that enable their moonlighting functions and then discussing the cellular processes and disease states that are influenced by these functions.     

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.Supplementary key words: low density lipoprotein receptor, receptors, protein trafficking, cholesterol, lipoproteins, CRISPR screen, HuH7 cells, endocytosis, RAB10, RAB11

An elevated level of circulating low-density lipoprotein (LDL) cholesterol is a major risk factor for atherosclerotic cardiovascular diseases, including myocardial infarction and stroke (1, 2, 3, 4, 5, 6, 7). Regulation of plasma cholesterol is governed by a complex interplay between dietary absorption, de novo biosynthesis, and clearance from the bloodstream. Therapeutic targeting of LDL clearance has been a highly successful strategy for the prevention and treatment of atherosclerosis. LDL clearance is mediated by the LDL receptor (LDLR), a cell-surface glycoprotein that directly binds to the apolipoprotein B component of LDL particles and triggers clathrin-mediated endocytosis. The acidic environment of the endosomal lumen induces complex dissociation, with LDL subsequently transported to the lysosome for hydrolysis, and free LDLR recycled back to the plasma membrane (8, 9). Many regulatory proteins affecting the endocytic pathway and cell-surface expression of LDLR have been identified, including PCSK9, a negative regulator that redirects LDLR to the lysosome for degradation (10), and IDOL, a ubiquitin ligase that induces proteasomal degradation of LDLR (11, 12). Although much is known about the regulation of LDLR expression and endocytosis, questions remain concerning the molecular determinants of LDLR recycling.We recently reported a genome-wide CRISPR screen for modifiers of LDL uptake in HuH7 cells (13). This screen identified RAB10, a small GTPase known to mediate trafficking of vesicles between intracellular compartments, as a key regulator of LDL uptake. Deletion of RAB10 decreased cellular endocytosis of LDL but increased accumulation of another endocytic cargo, transferrin. The receptors for LDL (LDLR) and transferrin receptor (TFR) are both endocytosed from the cell surface via clathrin-coated vesicles and transported through intracellular recycling pathways (14, 15, 16, 17, 18, 19, 20). In this study, we investigated the role of RAB10 in LDL and transferrin endocytosis. Our results demonstrate that GTP-bound RAB10 positively regulates the activity of LDLR and TFR by accelerating the recycling of both proteins to the plasma membrane.     

The Endolysosomal System in Cell Death and Survival

The endocytic pathway is a system specialized for the uptake of compounds from the cell microenvironment for their degradation. It contains an arsenal of hydrolases, including proteases, which are normally enclosed in membrane-bound organelles, but if released to the cytosol can initiate apoptosis signaling pathways. Endogenous and exogenous compounds have been identified that can mediate destabilization of lysosomal membranes, and it was shown that lysosomal proteases are not only able to initiate apoptotic signaling but can also amplify the apoptotic pathways initiated in other cellular compartments. The endocytic pathway also receives cargo destined for degradation via the autophagic pathway. By recycling energy and biosynthetic substrates, and by degrading damaged organelles and molecules, the endocytic system assists the autophagic system in resisting apoptotic stimuli. Steps leading to lysosomal membrane permeabilization and subsequent triggering of cell death as well as the therapeutic potential of intervention in lysosomal membrane permeabilization will be discussed.Since the discovery of lysosomes in 1950s (de Duve et al. 1955), the concept of the endocytic pathway has changed. Although there has been huge progress in understanding the molecular mechanisms of targeting and fusion of organelles, several conceptual dilemmas have not been completely resolved. The primary function of the endocytic pathway is bulk degradation and recycling of the internalized material and redundant cellular components. Over the years, additional functions have been associated with it. Endosomes and lysosomes can fuse with the plasma membrane to repair it and to release the accumulated nondegradable material (Medina et al. 2011). Intraluminal vesicles are the source of exosomes, which have multiple functions, especially for the immune system (Ludwig and Giebel 2012). Endosomes have numerous functions in fighting infections: they can signal the presence of pathogens through Toll-like receptors, they are the site of antigenic peptide generation and their assembly with major histocompatibility complex class II molecules, and they can also kill residing pathogens (Gruenberg and van der Goot 2006). Because of a high content of proteases, de Duve (1959) coined the figurative term “suicide bags” for lysosomes, a concept since supported by a wealth of experimental reports (de Duve 1959). Perhaps the best examples of this concept are natural killer cells and cytotoxic T cells. Both have specialized lysosome-related organelles, secretory granules, that contain perforin and granzyme B, which can mediate apoptosis in the target cell (Blott and Griffiths 2002; Trapani and Smyth 2002). However, every cell can potentially become a victim of its own lysosomal hydrolases, especially if lysosomal membranes are destabilized so that the enzymes can escape into the cytosol. These offer great potential to exploit scenarios for therapy for certain diseases, most importantly cancer. On the other hand, by enabling degradation of the material sequestered by autophagy, the endocytic pathway can assist autophagy in counteracting apoptosis when cells are challenged with an apoptotic stimulus (Repnik and Turk 2010; Hafner Česen et al. 2012; Repnik et al. 2012).     

Low density lipoprotein receptor-related protein 1 dependent endosomal trapping and recycling of apolipoprotein E

Background

Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling.

Principal Findings

Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion.

Conclusion

We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.     

Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

In mammalian cells, endocytosis plays a pivotal role in regulating several basic cellular functions. Up to now, the dynamics and the organization of the endocytic pathways have been primarily investigated in reductionist model systems such as cell and organ cultures. Although these experimental models have been fully successful in unraveling the endocytic machinery at a molecular level, our understanding of the regulation and the role of endocytosis in vivo has been limited. Recently, advancements in intravital microscopy have made it possible to extend imaging in live animals to subcellular structures, thus revealing new aspects of the molecular machineries regulating membrane trafficking that were not previously appreciated in vitro. Here, we focus on the use of intravital microscopy to study endocytosis in vivo, and discuss how this approach will allow addressing two fundamental questions: (1) how endocytic processes are organized in mammalian tissues, and (2) how they contribute to organ physiopathology.Endocytosis is a fundamental process used by the cell to internalize molecules from the plasma membrane (Mellman 1996; Doherty and McMahon 2009), and its dysregulation is the cause of several pathological conditions, such as cancer and neurodegenerative, metabolic, and storage diseases (Lanzetti and Di Fiore 2008; Mosesson et al. 2008; Ballabio and Gieselmann 2009).In mammals, endocytosis has been primarily studied in cell culture, which has been instrumental in identifying various endocytic pathways and elucidating the trafficking of internalized molecules throughout the endolysosomal system (Conner and Schmid 2003; Maxfield and McGraw 2004; Donaldson et al. 2009; Hurley and Stenmark 2011). The degree of complexity in the organization and the regulation of the endocytic processes have been shown to substantially increase in polarized cells (Mostov et al. 2003; Folsch et al. 2009) and in organ cultures (Dunn et al. 1980; Kandimalla et al. 2009; Khandelwal et al. 2010), which recapitulate some of the architectural features of the intact tissue. The scenario is further complicated in live animals, where tissues are continuously exposed to a specific combination of cues coming from the vasculature, the central nervous system, and the extracellular environment, which are difficult to reconstitute accurately in vitro. Therefore, although our knowledge of the molecular machineries controlling mammalian endocytosis has substantially increased in the last decades, there are still fundamental issues that have not been explored yet, such as how endocytic pathways are organized and regulated in mammalian tissues. Specifically, it is fundamental to establish whether in vivo cells show the same regulation of endocytic pathways that has been reported in vitro, or how molecules are internalized and trafficked in the presence of physiological levels of ligands and regulatory molecules. Another question is what is the contribution of the endocytic pathways to the physiopathology of a specific tissue or organ. For example, it is of paramount importance to determine whether and how endocytic pathways are altered in epithelial and stromal cells during tumor development and progression, and which specific cell function is affected by their dysregulation.Investigations of endocytosis in live mammals (i.e., rodents) were extensively performed during the 1980s and 1990s by using conventional techniques (e.g., biochemical assays, EM, and indirect immunofluorescence). However, the advent of the green fluorescent protein (GFP) technology, which has enabled imaging subcellular organelles in real time, has significantly shifted the focus toward cell cultures.The recent advancements in intravital microscopy (IVM), which encompasses a series of light microscopy–based techniques, have now made possible imaging biological processes in live animals at a subcellular resolution (Weigert et al. 2013). In this perspective, we focus on reviewing most of the recent data on IVM and endocytosis and try to convey to the reader a sense of the potential, challenges, and limitations of this approach. However, before discussing the “heart of the matter,” we start by briefly pointing out the advantages of using animal models versus the more popular and well-established in vitro model systems.     

Clathrin-Independent Pathways of Endocytosis

There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration.The identification of many of the components involved in clathrin-mediated endocytosis (CME) and their subsequent characterization have provided a window into how this complex process works. For example, understanding how a clathrin basket is assembled, and how adaptor complexes, the mechanochemical GTPase dynamin, and Rab GTPases work have given us insights into endocytic pit formation, cargo concentration, vesicle scission, and subsequent trafficking. These topics are described in detail elsewhere in this volume (see Johannes et al. 2014; Kirchhausen et al. 2014; Merrifield and Kaksonen 2014).Consequently, CME has remained a predominant paradigm for following the uptake of material into the cell. Several endocytic pathways that do not use clathrin and its attendant molecular machinery have begun to be recognized as distinct clathrin-independent endocytic pathways (CIEs) (see Fig. 1). Some of these pathways are constitutive, whereas others are triggered by specific signals or are even hijacked by pathogens. In addition, they differ in their mechanisms and kinetics of endocytic vesicle formation, associated molecular machinery, and cargo destination. Here we discuss characteristics of clathrin-independent (CI) endocytic pathways, the logic and mechanisms of cargo selection, vesicle budding, the itineraries of internalized cargo, and provide a perspective on the regulation of CIE.Open in a separate windowFigure 1.The diversity of endocytic pathways available at the cell surface of metazoan cells. The schematic outlines multiple means by which a cargo located at the plasma membrane or in the extracellular milieu enters the endocytic pathway in metazoan cells. Dynamin-dependent pathways (+; circles) are typically associated with small-scale coat-mediated invaginations, such as clathrin or caveolar pathways. The dynamin-independent pathways reflect a larger diversity of forms, ranging from the small-scale processes to the larger scale membrane invaginations. The main effectors of the CIE pathways are indicated below their primary invaginations. All the dynamin-independent mechanisms appear to use actin filament (red bars) polymerization machinery.At first glance CIE facilitates two types of endocytic processes—the large micrometer-scale pathways such as macropinocytosis and phagocytosis, and a spectrum of smaller (<200 nm) scale processes (Fig. 1). The large-scale processes involve internalization of significant patches of membrane, but these pathways may share some of the same molecular machinery as the smaller scale processes, especially those utilizing actin machinery in membrane remodeling, and have been addressed in recent reviews (Flannagan et al. 2012; Bohdanowicz and Grinstein 2013; see also Cossart and Helenius 2014).     

Cargo Recognition in Clathrin-Mediated Endocytosis

The endosomal system is expansive and complex, characterized by swift morphological transitions, dynamic remodeling of membrane constituents, and intracellular positioning changes. To properly navigate this ever-altering membrane labyrinth, transmembrane protein cargoes typically require specific sorting signals that are decoded by components of protein coats. The best-characterized sorting process within the endosomal system is the rapid internalization of select transmembrane proteins within clathrin-coated vesicles. Endocytic signals consist of linear motifs, conformational determinants, or covalent modifications in the cytosolic domains of transmembrane cargo. These signals are interpreted by a diverse set of clathrin-associated sorting proteins (CLASPs) that translocate from the cytosol to the inner face of the plasma membrane. Signal recognition by CLASPs is highly cooperative, involving additional interactions with phospholipids, Arf GTPases, other CLASPs, and clathrin, and is regulated by large conformational changes and covalent modifications. Related sorting events occur at other endosomal sorting stations.The internalization of a subset of plasma membrane proteins by clathrin-mediated endocytosis is one the best-characterized sorting processes that takes place in the endomembrane system of eukaryotic cells (Kirchhausen 2014). Selection of transmembrane proteins (referred to as “cargo”) for internalization by clathrin-mediated endocytosis involves recognition of endocytic signals in the cytosolic domains of the proteins by adaptors located in the inner layer of clathrin coats. Signal–adaptor interactions lead to concentration of the transmembrane proteins within clathrin-coated pits that eventually bud into the cytoplasm as clathrin-coated vesicles (Kirchhausen 2014). Transmembrane proteins that have endocytic signals are thus rapidly delivered to endosomes, whereas those that lack signals remain at the plasma membrane. This article summarizes recent progress in the elucidation of the mechanisms of signal recognition in clathrin-mediated endocytosis, with additional reference to related intracellular sorting events. Further information on this topic can be found in previous reviews (Bonifacino and Traub 2003; Traub 2009; Kelly and Owen 2011).     

Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Mediates Neuronal Aβ42 Uptake and Lysosomal Trafficking

Background

Alzheimer''s disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-β 42 (Aβ42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Aβ42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Aβ42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Aβ42 through LRP1 is lacking.

Methodology/Principal Findings

Here we show that LRP1 endocytic function is required for neuronal Aβ42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Aβ42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Aβ42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Aβ42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Aβ42 is associated with increased cellular toxicity.

Conclusions/Significance

These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Aβ42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Aβ metabolism.     

Endocytosis of Viruses and Bacteria

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own. However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination. The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology.Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles. This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.” In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010). With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such as Toxoplasma and Plasmodium (Robibaro et al. 2001).     

The Complex Ultrastructure of the Endolysosomal System

Live-cell imaging reveals the endolysosomal system as a complex and highly dynamic network of interacting compartments. Distinct types of endosomes are discerned by kinetic, molecular, and morphological criteria. Although none of these criteria, or combinations thereof, can capture the full complexity of the endolysosomal system, they are extremely useful for experimental purposes. Some membrane domain specializations and specific morphological characteristics can only be seen by ultrastructural analysis after preparation for electron microscopy (EM). Immuno-EM allows a further discrimination of seemingly identical compartments by their molecular makeup. In this review we provide an overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries.The endolysosomal network is required for multiple functions and control of cell homeostasis. It is not only reached by endocytic cargo but also by biosynthetic cargoes. It is an intermediate to degradation, but also essential for recycling, signaling, cell polarity, cilia formation, cytokinesis, and migration (Gould and Lippincott-Schwartz 2009; Taguchi 2013). This multitude of functions can only be ensured by an extremely organized ultrastructure. With the increased understanding of how cellular machinery defines endolysosomal subdomains, the nomenclature of the endolysosomal system has also increased in complexity. We start this review, therefore, with a brief introduction of the terminology of the endolysosomal system.Coated pits and vesicles were described in 1964 (Roth and Porter 1964), and lysosomes were first described by De Duve and Novikoff in the mid-1950s (Novikoff et al. 1956), but the range of organelles in between these beginning and ending stages of endocytosis was only described later (Bhisey and Freed 1971). Electron microscopy (EM) studies by Allen and coworkers on the unicellular ciliate Paramecium caudatum revealed the existence of intracellular compartments that could be loaded with the endocytic marker horseradish peroxidase (HRP) (Allen and Fok 1980). These were named “endosomes.” Parallel studies in mammalian cells, by Pastan, Willingham, and colleagues, also using HRP, described intracellular vacuoles and tubules involved in the transport of transferrin receptor (TfR) (Gonatas et al. 1977; Goud et al. 1981; Willingham and Pastan 1983). These were called “receptosomes” (Willingham and Pastan 1980). Geuze, Slot, and collaborators introduced immunogold labeling, allowing the quantitative localization of multiple proteins within one EM sample (Geuze et al. 1981). When they localized the recycling asialoglycoprotein receptor together with its ligand destined for lysosomal degradation (Geuze et al. 1983), they identified compartments consisting of a vacuole and multiple associated tubules. These were called compartments involved in the uncoupling of receptors and ligands (CURLs) because the vacuoles accumulated the ligand (for degradation) and the tubules the receptor (for recycling). Today the CURL is known as the “early endosome” (EE), which in addition to receptors and ligands is now known to be reached by virtually all components internalized from the cell surface (see Mayor et al. 2014; Cossart and Helenius 2014).In the current literature, different nomenclatures are still used to describe the endolysosomal system, which can sometimes cause some confusion. In this review, based on combined ultrastructural and functional knowledge, we propose the following nomenclature: We refer to the vacuolar domains of EEs as sorting endosomes (SEs) and the tubules emerging from SEs as recycling endosomes (REs). Although in some cells (e.g., melanocytes) (see Delevoye et al. 2009), the RE tubules may stay attached while functioning in recycling, more typically they detach from the SE to form a tubular endosomal network (TEN). The term “endosomal recycling compartment” (ERC) is used to designate the peri-centriolar compartment that can be observed only in some cell types. Late endosomes (LEs), also referred to as multivesicular bodies (MVBs), are rounded compartments filled with intraluminal vesicles (ILVs). Lysosomes are the final compartments of the endocytic pathway, with different morphologies depending on the cell type (schematic representation in Fig. 1). Moreover, in the literature, these terms are differently used because most studies involve light microscopy, which does not provide sufficient resolution to detect all of the distinct domains.Open in a separate windowFigure 1.Schematic and simplified representation of the endolysosomal system showing the different organelles described in this article. Sorting endosomes (SE) are vacuolar compartments often bearing bilayered, flat clathrin coats (brown). Tubules emanate from SE that form the recycling endosomes (RE). The RE may localize to the peri-Golgi area forming the endocytic recycling compartment (ERC) or distribute to the cell periphery. The RE network is complex with multiple sorting sites, thereby the tubular sorting endosome (TSE) or tubular endosomal network (TEN) is also represented. AP1 (red) and AP3 (green) coated buds on RE (ERC/TSE/TEN) are shown. Late endosomes correspond to multivesicular bodies (MVBs) filled with intraluminal vesicles (ILVs). MVBs are fated for fusion with lysosomes. In some cells, a population of MVBs fuse with the plasma membrane, a process during which the ILVs are secreted as exosomes. Gray arrows indicate directions of transport/maturation of compartments. Blue arrows indicate invagination at the endosomal membrane of SE and MVBs required for ILV formation.     

The endocytic network in plants

Endocytosis and vesicle recycling via secretory endosomes are essential for many processes in multicellular organisms. Recently, higher plants have provided useful experimental model systems to study these processes. Endocytosis and secretory endosomes in plants play crucial roles in polar tip growth, a process in which secretory and endocytic pathways are integrated closely. Plant endocytosis and endosomes are important for auxin-mediated cell-cell communication, gravitropic responses, stomatal movements, cytokinesis and cell wall morphogenesis. There is also evidence that F-actin is essential for endocytosis and that plant-specific myosin VIII is an endocytic motor in plants. Last, recent results indicate that the trans Golgi network in plants should be considered an integral part of the endocytic network.     

Lysosomal Lipid Storage Diseases

Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.Lysosomal lipid storage diseases are a group of inherited catabolic disorders in which typically large amounts of complex lipids accumulate in cells and tissues. Macromolecules such as complex lipids and oligosaccharides are constitutively degraded in the acidic compartments of the cell, the endosomes, and lysosomes, into their building blocks. The resulting catabolites are exported to the cytosol and reused in cellular metabolism. When lysosomal function is impaired because of a defect in a catabolic step, degradation cannot proceed normally and undegraded compounds accumulate. Lysosomal lipid storage diseases comprise mainly the sphingolipidoses, Niemann-Pick type C disease (NPC), and Wolman disease, including the less severe form of this disease, called cholesteryl ester storage. NPC is a complex lipid storage disease mainly characterized by the accumulation of unesterified cholesterol in the late endosomal/lysosomal compartment (Bi and Liao 2010). The sphingolipidoses are caused by defects in genes encoding proteins involved in the lysosomal degradation of sphingolipids (Kolter and Sandhoff 2006). First reports on these diseases were given more than a century ago. Already in 1881, Warren Tay described the clinical symptoms of a disease, which is today called Tay-Sachs disease (Tay 1881). After Christian de Duve discovered the lysosome in 1955 (de Duve 2005), Henri-Géry Hers established the first correlation between an enzyme deficiency and a lysosomal storage disorder (Pompe’s disease) in 1963 (Hers 1963). In the following decades, the enzymes and cofactors deficient in the sphingolipidoses have been identified. Though lysosomal lipid storage diseases have been known for a long time, treatment is only available for a few mild forms of the diseases, such as the adult forms of Gaucher disease (Barton et al. 1991). For several lysosomal storage diseases, therapies like enzyme replacement or bone marrow transplantation are in the clinical trial stage (Platt and Lachmann 2009). For a long time, lysosomal diseases have been considered a problem of superabundance (storage) in which the storage material can slowly spread to other cellular membranes, impairing their function. More recently, it came into focus that massive storage prevents lysosomal functions such as nutrition delivery through the endolysosomal system, leading to a state of cellular starvation. In mouse models of both GM1 and GM2 gangliosidoses iron is progressively depleted in brain tissue. Administration of iron prolonged survival in the diseased mice by up to 38% (Jeyakumar et al. 2009).     

Neuronal Signaling through Endocytosis

The distinctive morphology of neurons, with complex dendritic arbors and extensive axons, presents spatial challenges for intracellular signal transduction. The endosomal system provides mechanisms that enable signaling molecules initiated by extracellular cues to be trafficked throughout the expanse of the neuron, allowing intracellular signals to be sustained over long distances. Therefore endosomes are critical for many aspects of neuronal signaling that regulate cell survival, axonal growth and guidance, dendritic branching, and cell migration. An intriguing characteristic of neuronal signal transduction is that endosomal trafficking enables physiological responses that vary based on the subcellular location of signal initiation. In this review, we will discuss the specialized mechanisms and the functional significance of endosomal signaling in neurons, both during normal development and in disease.Endocytosis is a basic cellular process that has been conserved and adapted from single cell eukaryotes through humans (reviewed in Mellman 1996; Mukherjee et al. 1997). The fundamentals of endosomal recycling and degradation are the same in neurons as in other cell types (reviewed in Yap and Winckler 2012). However, the endocytic machinery is particularly important in neurons, as specialized vesicles are engaged in releasing neurotransmitters and in subsequent membrane retrieval (reviewed in Saheki and De Camilli 2012; von Zastrow and Williams 2012). Furthermore, endocytosis of neuronal growth factor receptors regulates where and when signaling cascades are initiated (reviewed in Hupalowska and Miaczynska 2012). Here we will discuss how the endocytic process in neurons is adapted so that vesicles can travel through the extensive span of neuronal axons and dendrites, and convey spatial information.     

Endocytosis and Signaling during Development

The development of multicellular organisms relies on an intricate choreography of intercellular communication events that pattern the embryo and coordinate the formation of tissues and organs. It is therefore not surprising that developmental biology, especially using genetic model organisms, has contributed significantly to the discovery and functional dissection of the associated signal-transduction cascades. At the same time, biophysical, biochemical, and cell biological approaches have provided us with insights into the underlying cell biological machinery. Here we focus on how endocytic trafficking of signaling components (e.g., ligands or receptors) controls the generation, propagation, modulation, reception, and interpretation of developmental signals. A comprehensive enumeration of the links between endocytosis and signal transduction would exceed the limits of this review. We will instead use examples from different developmental pathways to conceptually illustrate the various functions provided by endocytic processes during key steps of intercellular signaling.The evolution of multicellular life introduced a division of labor between specialized cells, which strongly increased demand for intercellular communication both during development and homeostasis of the adult organism (Kaiser 2001). At the genomic level this is reflected by a dramatic expansion of the surface receptor signalome in all metazoan lineages (Ben-Shlomo et al. 2003). However, the idea that intercellular communication drives the organization and patterning of the embryo precedes the identification of the responsible molecules. Induction (i.e., the ability of one group of cells within a developing organism to influence the cell-fate choices), morphogenesis, and differentiation of other cell populations, was firmly established by the experiments of Spemann and Mangold (1924) and has become one of the most important concepts of developmental biology. The related concept of the morphogen, whereby a cell can identify its position within a tissue by using the local levels of a secreted molecule forming a concentration gradient as a proxy for its distance from the source, was famously illustrated by the “French flag Model” by Wolpert (1969).Since then, examples of developmental patterning events following these two paradigms have been identified in all developmental model systems, ranging from worms and flies to amphibians, fish, and mice, and even humans. Surprisingly, despite the huge variety in the eventual outcome of metazoan embryonic development, it turned out that most individual patterning events are performed by a restricted set of signal-transduction pathways that are used repeatedly and in varying cellular contexts (Pires-daSilva and Sommer 2003; Perrimon et al. 2012).Because animal embryos differ in shape and size, closely related pathways must function over similarly varying spatial and temporal scales, potentially even at successive developmental stages within the same embryo. To understand how the limited, intercellular signaling repertoire is modulated to accommodate the varying patterning needs arising within the different developmental contexts, it is necessary to study the signal-transduction machinery at the molecular level. In recent years, major progress has been made in understanding the mechanistic links between the protein trafficking machinery and the generation and interpretation of morphogenetic signals.Traditionally, endocytosis was seen as a means of removing activated receptors and their bound ligands from the surface of the signal-receiving cells, thereby terminating the signals. However, positive effects of endocytosis on signal transduction have recently been identified for many different pathways including, among others, receptor tyrosine kinases (RTKs), TGF-β, TNF-α, Toll-like receptor, Wnt, and Notch signal-transduction cascades (Miaczynska et al. 2004; Platta and Stenmark 2011). In many of these examples, endosomes act as platforms where the activated receptors can interact with specific downstream components of the signal-transduction machinery (Sadowski et al. 2009; Miaczynska and Bar-Sagi 2010). Trafficking of the receptors into and out of such endosomes may thus provide another tier for the regulation of the signaling output that allows temporal and spatial modulation of the signals independent of ligand presentation. In addition, the endocytic pathway has recently also become implicated in signaling events that precede the intracellular transduction of the signal. In this review, we therefore focus on how the endocytic machinery participates in the generation, propagation, reception, and interpretation of intercellular signals in the context of animal development.     

Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery.At the cellular level, receptor tyrosine kinases (RTKs) need to be properly localized to function as signal-receiving and signal-transmitting devices (Lemmon and Schlessinger 2010). To receive signals (i.e., to bind extracellular ligands), RTKs have to be exposed at the surface of the plasma membrane. To transmit signals after ligand binding by RTKs, appropriate signaling components have to be available within intracellular compartments: in the cytoplasm, in association with membrane-bound organelles and in the cell nucleus. Importantly, the intracellular distribution of RTKs and their associated partners is not static but undergoes dynamic changes in different phases of signaling, as reflected for example by endocytic internalization of activated RTKs (Scita and Di Fiore 2010). Therefore, to function properly, the whole RTK signaling machinery within the cell has to be organized and tightly controlled both in space and in time. This organization and control are ensured by intracellular trafficking machineries, mainly by membrane transport systems such as endocytosis and secretion but also by other distribution systems (e.g., responsible for nucleocytoplasmic shuttling of proteins).Recent years have brought increasing evidence that intracellular membrane trafficking, in particular endocytic internalization, degradation, and recycling, can profoundly affect the signaling properties of RTKs (Mukherjee et al. 2006; Abella and Park 2009; Lemmon and Schlessinger 2010; Scita and Di Fiore 2010; Grecco et al. 2011; Sigismund et al. 2012). The changes in the amounts of RTKs at the cell surface can alter the cellular responses when ligands are abundant (Grecco et al. 2011). In turn, the presence of a given RTK at the plasma membrane is determined by the rates of three trafficking processes: delivery of newly synthesized molecules by the secretory pathway, their internalization (occurring for both ligand-bound and ligand-free molecules), and endocytic recycling. Although the molecular details concerning the regulation of RTK delivery to the plasma membrane are not well known, numerous studies document various mechanisms by which internalization and recycling of RTKs can be modulated, thus affecting the signaling outputs (Le Roy and Wrana 2005). In addition to the regulation of RTKs at the cell surface, trafficking processes control the intracellular fate of endocytosed RTKs. Following internalization, RTKs can be either targeted for lysosomal degradation, or recycled back to the plasma membrane (Mukherjee et al. 2006; Abella and Park 2009; Scita and Di Fiore 2010). The first route results in the termination of signaling, whereas the second allows for sustained signaling if the ligand is available. Usually degradation and recycling of a given RTK can occur simultaneously but the balance between them is crucial to determine the net signaling output. Again, the molecular mechanisms that can shift the fate of internalized RTKs between degradation and recycling, thus changing RTK signaling, have begun to emerge in recent years (Polo and Di Fiore 2006; von Zastrow and Sorkin 2007; Sorkin and von Zastrow 2009; Sigismund et al. 2012). Finally, in contrast to an early view that only RTKs present at the plasma membrane are signaling competent, it is now accepted that in many cases activated RTKs can emit signals also after internalization into intracellular compartments (Miaczynska et al. 2004b; Miaczynska and Bar-Sagi 2010; Platta and Stenmark 2011). In some cell types (e.g., in neurons), such “signaling endosomes” are crucial for signal propagation within the cell and for the final cellular response. Moreover, endosomes can serve as platforms for amplification and compartmentalization of signals emitted by RTKs (Sadowski et al. 2009; Platta and Stenmark 2011).In this article, I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. As the membrane trafficking system of a cell is highly interconnected and can be considered a global dynamic continuum, it is important to note that often one primary alteration at a given stage of RTK trafficking may affect other transport steps or compartments, thus causing generalized changes in the intracellular routing and signaling of RTKs.