© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12147
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Systems Dynamics in Endocytosis Maximilian Fürthauer1∗ and Elizabeth Smythe2∗ 1 Institut 2 ∗
de Biologie Valrose, CNRS UMR7277, INSERM 1091, University of Nice Sophia-Antipolis, Nice, France Centre for Membrane Interactions and Dynamics, Department of Biomedical Science, University of Sheffield, Sheffield, UK
Corresponding authors: Maximilian Fürthauer, [email protected] and Elizabeth Smythe, [email protected]
Abstract The endocytic system acts at the crossroads of different cellular activities
to complement the insights that can be gained by biochemical and cell
to play a central role in the regulation of cell signaling and membrane
biological approaches with the use of quantitative biophysics, systems
dynamics. An European Molecular Biology Organization (EMBO) confer-
biology and animal model systems to achieve an integrated view of
ence held in October 2013 in Villars-sur-Ollon gathered researchers from
the properties of the endomembrane system and its role in cellular
all over the world to present their latest findings on the endolysosomal
information processing.
system and identify major challenges for the future. The conference covered the entire spectrum of research in this rapidly evolving field
Keywords endocytosis, lipids, membrane dynamics, signaling, systems biology
ranging from the cellular mechanics of endocytosis to the role of proteins and lipids in the biogenesis and function of endolysosomal
Received 10 December 2013, revised and accepted for publication 22
organelles and the analysis of higher order system properties in multi-
December 2013, uncorrected manuscript published online 9 January
cellular contexts. In particular, the meeting highlighted current efforts
2014
The year 2013 saw the passing of Christian de Duve, the discoverer of lysosomes and 1974 Nobel Prize in Physiology and Medicine winner. In 1963, de Duve coined the term ‘endocytosis’ to designate a process in which a plasma membrane invagination leads to the formation of intracellular carrier vesicles that allow the engulfment of particles or fluids from the extracellular environment (1). An EMBO conference organized by Marcos GonzálezGaitán (University of Geneva, Switzerland) and Ludger Johannes (Institut Curie, Paris, France) on the ‘Systems Dynamics of Endocytosis’ provided ample evidence that 50 years after de Duve’s naming, research in the field of endocytosis is more vibrant than ever.
the plasma membrane, the cytoplasmic organelles as well as biosynthetic and degradative compartments. The endolysosomal system has emerged as a central regulator of cellular activities that govern not only nutrient uptake and signal interpretation from the cell surface, but also the regulation of cellular metabolism and the capacity of a cell to communicate with its neighbors. It is therefore no surprise that dysfunctions of this system have been shown to be highly deleterious to animal development and linked to a number of human diseases. The conference presentations reflected this breadth.
The Cellular Mechanics of Endocytosis Five decades of research have indeed revealed that the importance of endocytic trafficking extends far beyond the mere uptake of materials from the extracellular space. The paramount importance of this system is due to its ability to serve as a cellular highway that interconnects traffic between different cellular compartments including 338 www.traffic.dk
Where it all starts: the growing diversity of endocytic entry portals Clathrin-dependent endocytosis While clathrin-mediated endocytosis (CME) is undoubtedly the most studied and best understood endocytic
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entry route, a number of lively discussions highlighted the controversies that still surround this complex process. However, the discussions also revealed that a consensus is emerging with respect to key aspects of clathrin-coated pit (CCP) and vesicle (CCV) formation. Live cell imaging is unraveling the dynamics of CCP formation at the plasma membrane, demonstrating that it is a stochastic process. Clathrin assemblies can initiate and then disassemble (abort) very rapidly. Tom Kirchhausen (Harvard Medical School, Boston, MA, USA) described how CCP initiation requires the cooperative use of weak interactions between the plasma membrane phospholipid PI(4,5)P2 , the endocytic adapter protein AP2 and clathrin. Total Internal Reflection Fluorescence (TIRF) microscopy with single molecule sensitivity has determined that a stoichiometry of 2 AP2 molecules per clathrin triskelion is required for the initiation of CCP formation but not sufficient to guarantee further CCP to CCV maturation (2), (Figure 1). While early CCPs may still undergo disassembly, Kirchhausen proposed that gatekeepers such as FCHo proteins and cargo stabilise the maturing pit leading to productive CCV formation (3). Sandy Schmid (UT Southwestern, Dallas, TX, USA) extended the quantitative analysis of CME to include CCP maturation, that precedes dynamin-dependent scission to form a CCV (4). Her study revealed that many pits also abort relatively late (up to 20 seconds in a 60 second time-scale), suggesting the existence of specific checkpoints, analogous to the gatekeepers discussed above, which regulate maturation. Dynamin recruitment is key to functional CCV formation and so may be one such checkpoint. Consistent with previous studies (5), dynamin was observed to be recruited early in CCP assembly. A second checkpoint involves the diverse family of endocytic accessory proteins (EAPs) which contribute to recruitment of specific cargo and curvature generation at CCPs. Analysis of an AP2 mutant that is impaired for EAP binding revealed a surprising robustness of CME: while a failure in EAP recruitment resulted in an increased frequency of abortive CCP assembly events, this defect was compensated for by an increase in the overall number of initiation events and the speed of CCP/CCV maturation, thus explaining why endocytosis of housekeeping cargo such as transferrin receptor is unaffected in the absence Traffic 2014; 15: 338–346
Figure 1: Clathrin coated pit assembly. The cartoon shows how two AP2 molecules tether a single clathrin triskelion to the inner surface of the plasma membrane in the initial stages of clathrin lattice assembly. As more AP2 complexes and clathrin assemble, there is increased membrane curvature until scission results in a coated vesicle. Courtesy of Tom Kirchhausen, Harvard University. of EAP binding (6). The development of sensitive assays to monitor CCP dynamics in the context of individual pits containing specific cargo will greatly facilitate our understanding of the roles of EAPs. Expanding on the theme of cargo determining coated pit dynamics, Liz Smythe (University of Sheffield, UK) described how clathrin light chain phosphorylation helps to define subpopulations of CCPs containing different G protein-coupled receptors and how this segregation may impact on downstream signaling (7). Both Kirchhausen and Schmid emphasized the methodological challenges associated with quantitative live cell imaging and the necessity to have unambiguous definitions, e.g. the criteria used to define productive versus 339
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abortive CME events. There was a discussion of the relative merits of overexpression of endocytic proteins versus genome-edited cells that express functional green fluorescent protein (GFP)-fusion proteins at endogenous levels which has been heralded as the gold standard for the future (8). This would apply especially to EAPs which compete with each other for common AP2 binding sites and should therefore not be overexpressed. Sandy Schmid highlighted that the intrinsically low fluorescence obtained in genome-edited cells may prevent the proper tracking and interpretation of labeled structures. For example, previous claims that overexpression of tagged clathrin light chains impairs CME (8) are likely to reflect inaccuracies in CCP tracking due to low signal-to-noise ratios. Having a fiduciary marker of high signal-to-noise ratio substantially improves data analysis and interpretation (4). A key element to facilitate understanding of the complexity of the endocytic system from the molecular through to the organismal level will be the availability of well-characterized toolboxes to systematically investigate protein function. Tom Kirchhausen initiated a discussion on the potential to establish a resource of genome-edited mammalian cells expressing key components of the endocytic pathway which would then provide a valuable and robust experimental system for the entire community. Aur´elien Roux (University of Geneva, Switzerland) presented a radically different approach to understanding how the biophysical properties of membranes contribute to membrane scission. His studies used purified components to reconstitute individual endocytic events in vitro to reveal that membrane scission depends on membrane tension and bending rigidity as well as on the torque of the constricting dynamin helix (9). A local change in membrane curvature at the edge of the dynamin helix results in a local variation in line tension and membrane elastic energy that determines the location of the scission event. Extending this type of approach to the analysis of CME can generate a biophysical phase diagram that describes the transition from an initial flat sheet of membrane, through a shallow CCP and toward the mature CCV depending on the local biophysical properties of the system. The importance of the biophysical properties of the membrane is supported by differing requirements for CME depending on context. In this regard a consensus 340
is emerging that actin, the involvement of which in mammalian endocytosis has long been debated, is required in environments with elevated membrane tension (10) such as the rigid cholesterol/sphingolipid-rich apical surface of epithelial cells as opposed to the more floppy basolateral membrane. Clathrin links to actin via interactions of clathrin light chains with Hip1 and Hip1R, actin binding proteins. Frances Brodsky (UCSF, USA) discussed how clathrin function is surprisingly diverse and extends beyond classical CCV formation (11). One example of this is clathrin-dependent recruitment of actin which facilitates internalization of bacteria via the clathrin light chain-Hip pathway (12). A challenge for the future is imaging of clathrin dynamics in vivo in vertebrate systems. Tom Kirchausen showed some intriguing imaging using both whole cell live cell imaging and intra vital microscopy indicating that such approaches are not far off.
Clathrin-independent endocytosis In addition to CME, cells use a wide variety of alternative, clathrin-independent endocytosis (CIE) pathways, whose inter-relationships and regulation is similarly complex. Nathalie Sauvonnet (Institut Pasteur, Paris, France) presented novel insights into the trafficking of the Interleukin 2 cytokine Receptor (IL-2R) which is internalized via a CIE pathway that requires Dynamin, Phosphatidylinositol-3-Kinase and an actin-regulator NWASP (13). Ultrastructural analysis of cells undergoing IL-2R endocytosis suggests that this process may involve protrusions emerging from the cell surface. Synaptic vesicle recycling endocytosis has to occur on a much faster time-scale (within a few seconds) than classical CME. Harvey McMahon (MRC-LMB, Cambridge, UK) presented evidence for the existence of a novel CIE pathway that might fulfill these kinetic requirements and which, while active in non-neuronal cells, is likely to be essential in neurons. A major technical issue for the future will be to achieve a direct visualization of fast endocytic events in the physiological context of the neuronal synapse. As for CME, pathways for CIE have been uncovered through the analysis of pathogenic toxins that induce their own endocytosis. Ludger Johannes described the Traffic 2014; 15: 338–346
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capacity of the B-Subunit of Shiga Toxin to bind the glycosphingolipid Gb3 at the cell surface, generate membrane curvature and trigger actin-dependent scission of endocytic carrier vesicles (14). For the Shiga toxin uptake pathway, his group has now identified a BAR domain protein that is required not for initial membrane bending (that is driven by the toxin), but for subsequent scission in synergy with actin. Other recent data suggest that glycosphingolipids also play a role in physiological, non-pathogenic endocytosis of endogenous cargo proteins such as CD44, and that this process is driven by endogenous lectins by mechanisms similar to the one discovered for Shiga toxin. While self-induced endocytosis of bacterial toxins is an essential first step for entry into host cells, toxins must ultimately leave their endocytic vehicle to enter the cytoplasm and exert their effect on host cell physiology. Gisou van der Goot (EPFL, Lausanne, Switzerland) presented the case of the anthrax toxin lethal factor (LF) which takes advantage of the pore-forming protective antigen to translocate into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs). Back-fusion of ILVs with the endosomal limiting membrane is then proposed to release LF into the cytoplasm. New data reveal that MVEs do not only ensure immediate LF release into the cytoplasm but that they can also act as long term toxin reservoirs, the content of which can be released by exosomes into the extracellular medium even after the bacteria themselves have been cleared from the organism (15). Intriguingly, this may indicate either the existence of a subpopulation of stable late endosomal compartments or the presence of both short and long lived ILVs within a common MVE. Membrane microdomains: from endocytosis to stress response and cell signaling Rob Parton (IMB, Brisbane, Australia) illustrated that internalization of plasma membrane components is not always linked to cargo uptake. While endocytosis of caveolae can account for a substantial fraction of total cellular endocytic uptake under certain circumstances, it is becoming evident that the primary function of caveolar endocytosis is to regulate their own abundance at the cell surface (16). In this context cells can respond to mechanical stress by caveolar disassembly as a protective mechanism to ensure membrane integrity (17), an Traffic 2014; 15: 338–346
observation that may shed novel light on muscular dystrophies resulting from human caveolar dysfunction. Interestingly membrane stretching disrupts the interactions between caveolins and their essential cavin partner proteins. As cavins have initially been described as nuclear proteins this begs the question whether the membrane release and nuclear translocation of these molecules may trigger stress-induced signaling. By combining biochemical analyses, mass spectrometry and high resolution electron microscopy Benjamin Nichols (MRC-LMB, Cambridge, UK) provided evidence that caveolae are composed of repeated units of a caveolar coat complex composed of cavins and caveolins (18). Knockout mice for cavin2 and cavin3 reveal that despite this unifying architecture, deletion of individual cavin genes results in tissue-specific phenotypes that are indicative of caveolar heterogeneity (19). While the main role of caveolae appears to be as a structural membrane organizer, membrane microdomains have long been proposed to play an essential role in the dynamic assembly of cellular signaling platforms. Experimental support to substantiate this attractive hypothesis has however been difficult to obtain. Christophe Lamaze (Institut Curie, Paris, France) presented evidence for an essential role of plasma membrane rafts in the regulation of cell signaling by the Interferon Gamma receptor (20). Structural analysis of the endocytic machinery While live imaging and functional assays have yielded a wealth of information with respect to the roles and hierarchy of individual endocytic proteins, a major challenge is to determine at the molecular level how multiprotein machines assemble. Two studies illustrated the use of latest-generation electron microscopy to bridge the gap between the structure and the function of the endocytic machinery. Marko Kaksonen (EMBL, Heidelberg, Germany) presented an elegant combination of correlative electron tomography (20) and single particle tracking to determine the dynamics of recruitment of endocytic proteins during CME in yeast. The power of this approach is highlighted by its capacity to measure the number of molecules involved in a given event as well as providing information about the 341
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100 nm
100 nm
Figure 2: Correlative electron microscopy is a powerful tool to understand how endocytic proteins contribute to CCP structure. The left panel shows a centroid trajectory of a GFP-tagged yeast endocytic coat protein patch during vesicle budding. The right panel shows an endocytic membrane invagination identified by fluorescence microscopy and then imaged at high resolution by electron tomography. Courtesy of Andrea Picco, Wanda Kukulsk and Marko Kaksonen (EMBL). spatial orientation of a molecule during macromolecular assembly (Figure 2). Using in vitro assembly of purified protein components on defined lipid templates, Scott Emr (Cornell University, Ithaca, NY, USA) provided structural information about the assembly of the ESCRTIII machinery that forms membrane-shaping helices required for membrane scission during the biogenesis of ILVs of MVEs, cytokinetic abscission and viral budding. While oligomerization of the major ESCRTIII component SNF7/CHMP4 is sufficient for the formation of flat spirals on lipid monolayers, incorporation of Vps24 and Vps2 triggers the transformation of these 2-dimensional spirals into 3dimensional helices that may act to deform associated membranes (21). Combined assembly of ESCRTII and III subcomplexes leads to the formation of a macromolecular ring that may confine membrane and the cargo content of a single ILV (22).
Biogenesis, Function and Connectivity of Endolysosomal Organelles Lipids end endocytosis While technical limitations have for a long time confined the vast majority of cell biological studies to the analysis of protein components, it is becoming clear that a comprehensive understanding of the endolysosomal 342
system will depend on our ability to characterize the functional contribution of the many lipid species that compose different cellular membranes. Cellular cholesterol is mostly derived from the endocytic uptake of low density lipoproteins and their subsequent de-esterification and mobilization from late endosomal compartments. Jean Gruenberg (University of Geneva, Switzerland) illustrated the importance of the late endosomal phospholipid lysobisphosphatidic acid (LBPA) for the export of endocytosed cholesterol through a poorly understood trafficking route that may involve the back-fusion of ILVs with the late endosomal limiting membrane, a process that may be mechanistically related to the endosomal egress route used by Vesicular Stomatitis Virus (23). In support of this hypothesis, functional screens for the regulation of endosomal lipid composition have identified cellular factors that are required for both processes. The impact of lipid composition on endosomal trafficking was illustrated by Kirsten Sandvig (University of Oslo, Norway). Lipidomic analysis of cells grown at various densities reveals major changes in different lipid species (24) which correlated with significant changes in the cell surface binding and retrograde transport of Shiga toxin. Different cell types differ in their density-dependent regulation of Shiga toxin receptor levels but not for the density-dependence of Shiga toxin binding to the cell surface. These observations suggest that toxin/receptor Traffic 2014; 15: 338–346
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binding is not governed by abundance only but also by the lipid environment of receptor molecules. In the course of an RNAi-based screen designed to identify novel regulators of CME, Margaret Robinson (CIMR, Cambridge, UK) discovered an unexpected link between the vacuolar V-ATPase and the regulation of plasma membrane cholesterol (25). Ultrastructural analysis revealed the presence of non-constricted clathrin coated invaginations at the surface of V-ATPase depleted cells. The observation that the appearance of these aberrant structures can be rescued by cholesterol addition suggests that V-ATPase activity may be required for the transport of cholesterol from endosomes to the plasma membrane. Sergio Grinstein (Hospital for Sick Children, Toronto, Canada) illustrated the use of a fluorescent probe based on the lactadherin C2 domain to follow the cellular dynamics of phosphatidylserine (PS), a major component of the inner plasma membrane leaflet (26). A functional screen of nonessential genes in the yeast genome led to the discovery that ergosterol (the yeast equivalent of cholesterol) is important for PS distribution. This effect on PS can be explained by the importance of cholesterol for endosomal recycling, highlighting the fact that endocytic trafficking is not only lipid-dependent but also essential to maintain the cellular distribution of the lipids themselves. Biogenesis and functional connections of endolysosomal organelles Anne Spang (Biozentrum, Basel, Switzerland) described mechanisms by which one organelle can be converted to another by studying the conversion of Rab5-positive early endosomes into Rab7-positive late endosomes (27) and the role of HOPS and Corvet complexes. The macrophagelike coelomocytes of the nematode Caenorhabditis elegans present a natural population of gigantic endosomes that allow visualization of endosomal maturation processes using light microscopy. A major question for future studies will be to determine whether endosome conversion is regulated by the biophysical properties of the system (e.g. phosphoinositide composition) or the direct monitoring of organelle size. Two presentations illustrated how different cellular organelles can communicate through the use of specialized Traffic 2014; 15: 338–346
contact sites. Using a combination of imaging approaches from electron microscopy to optogenetic manipulation of cellular phosphoinositide levels, Francesca Giordano (De Camilli Lab, Yale School of Medicine, New Haven, CT, USA) provided evidence for the existence of contact sites, mediated by extended synaptotagmins, between the endoplasmic reticulum (ER) and the plasma membrane, that are functionally distinct from previously characterized contact sites regulated by STIM1 and Ora1 (28). While functional connections between the endosomal system and the Golgi apparatus have long been known, it has only recently become evident that specialized contact zones also exist between endosomes and the ER (29). Harald Stenmark (University of Oslo, Norway) presented evidence for the importance of ER-endosome contact sites in the regulation of endosome motility as vesicles switch from an initial centripetal to a later centrifugal movement through the recruitment of specific motor proteins. Cells modulate the size and composition of their organelles in response to environmental conditions. Andreas Mayer (University of Lausanne, Switzerland) raised the question of whether the content of an organelle influences its size? The size of the yeast vacuole is regulated in response to nutrient availability (30). A single molecular species, inorganic poly-phosphate accounts for the vast majority of vacuolar content. Consequently manipulations of polyphosphate synthesis induce significant alterations in vacuolar content and impact on organelle size and number. It will be of interest to know the relative contributions of intrinsic physicochemical properties and extrinsic sensing mechanisms in the control of organelle size.
Integrative Approaches for the Analysis of the Endolysosomal System Systems biology of endocytosis Beyond the mechanistic characterization of individual endocytic events, a number of presentations highlighted current efforts to achieve an understanding of endomembrane system using systems biology approaches. Marino Zerial (MPI-CBG, Dresden, Germany) presented studies on the higher order properties of the endocytic system with respect to the regulation of cellular signal transduction (31). Through a ground-breaking quantitative 343
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analysis of endosomal signal transduction he provided evidence that the endocytic system acts as a cellular information processing centre that converts incoming external stimuli into digitalized biological information. Interestingly, differences in this cellular information processing can account for the capacity of different growth factors to generate different biological responses while sharing common downstream components. Pier Paolo Di Fiore (IFOM, Milan, Italy) raised the question of how the wiring of the endocytic system enables it to accommodate the wide range of EGF concentrations that are encountered in vivo. Low EGF concentrations induce CME and receptor recycling, while high concentrations induce ubiquitination-dependent CIE, and subsequent receptor degradation (32). The threshold-controlled ubiquitination that is required for CIE converts a gradient of increasing EGF concentration into a threshold-activated ON/OFF response. A mathematical model of EGF/EGFR endocytosis describes how this behavior can account for the capacity of the system to cope with a wide range of ligand concentrations which can differ over two orders of magnitude in normal physiological conditions. Strikingly, the model reveals that system robustness is observed only within certain limits and that these may be surpassed by the extremely high EGF concentrations present in cancerous tissues.
of cell–cell interactions. Lukas Pelkmans (University of Zürich, Switzerland) approached the influence of cell density on the properties of the endocytic system. The use of a newly established approach for imagebased transcriptomics (35) combines information about the crowding status of individual cells with their transcriptional response. The use of this methodology permits the identification of the factors involved in the sensing of cell crowding as well as the downstream mediators of the cellular crowding response. It is particularly striking to note that this type of analysis reveals how social behavior of cells can be generated in a purely cell-intrinsic way, without the necessity for dedicated pathways of cell-to-cell communication. From cells to tissues: endocytic trafficking in multicellular organisms One of the most obvious characteristics of multicellular organisms is the organization of cells into polarized tissues of defined spatial orientation. Four presentations dealt with the impact of cell and tissue polarity on the regulation of the cellular endomembrane system. Through a large-scale analysis of key regulators of cellular membrane trafficking in different fly tissues Susanne Eaton (MPI-CBG, Dresden, Germany) revealed that there is no universal relationship between cell polarity and the positioning of endolysosomal organelles in polarized tissues. Rather than being stereotypes, the positioning of organelles appears to be highly variable and adapted to the specific requirements of a given cellular context.
Following de Duve’s pioneering discovery of lysosomes it has become clear that this organelle is more than simply a cellular garbage disposal unit since it also plays essential roles in homeostasis through the regulation of protein degradation, autophagy and lysosomal exocytosis. Lysosomal adaptation posits that the entire spectrum of functions of an organelle is adjusted to the demands of cellular physiology by a central master regulator. Taking advantage of a systems biology approach, Andrea Ballabio (TIGEM, Naples, Italy) presented the transcription factor TFEB as a master regulator of lysosomal activities. The potency of this master regulator is further illustrated by the striking observation that the overexpression of TFEB rescues lysosomal storage disorders (33) and enhances lipid degradation in mouse models of obesity (34).
Accordingly, Emmanuel Derivery from the lab of Marcos González-Gaitán (University of Geneva, Switzerland) described the mechanisms through which the polarization of an asymmetrically dividing Drosophila sensory precursor cells drives the directional movement of a population of signaling endosomes that can be identified through the presence of the endosomal adapter protein SARA and ensure the transport of Delta ligands and their cognate Notch receptors (36). Careful quantitative analysis of individual endosome motility in high resolution time lapse recording reveals the role of the polarized cytoskeleton in the regulation of asymmetric endosome segregation.
One of the fundamental characteristics of living organisms is the variability of their constituent cells and the roles
Gillian Griffiths (CIMR, Cambridge, UK) described the regulation of polarized exocytosis at the immune synapse.
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In this context cellular polarity is not governed by the overall organization of the tissue but rather by the site of interaction between cytotoxic T-cells and antigenpresenting cells. Following activation, T-cells promote the biogenesis of secretory lysosomes. The polarized delivery of these cytotoxic organelles is determined by the directional movement of the T-cell centrosome toward the immunological synapse (the specialized contact zone between a T-cell and antigen-presenting cell) and polarization of the cytoskeleton. Most interestingly, a thorough cell biological analysis of activated T-cell behavior suggests that the immunological synapse may share important properties with primary cilia, including a role for hedgehog (Hh) signaling in T lymphocytes. However, in contrast to Hh signaling in the cilium, Hh signaling is mediated by an intracellular pool of Indian Hh which colocalizes with the its receptor (Patched) on a subset of endosomes (37). Jiˇrí Friml (ISTA, Klosterneuburg, Austria) illustrated the importance of polarized endocytic trafficking for the generation of directional gradients of the plant hormone Auxin that play a major role in plant morphogenesis. Directional Auxin transport depends on the polarized localization of Pin-formed (PIN) auxin transporters that can be regulated by intrinsic developmental and extrinsic environmental cues (38). PIN localization is regulated through continuous CME and endosomal recycling that ensures rapid shuttling of molecules between endosomes and the different polar domains at the plasma membrane. A computer model that integrates the dynamics of PIN trafficking with feedback regulation by auxin can fully account for the role of this major plant hormone in plant embryonic development. Altogether, the EMBO conference on ‘Systems Dynamics of Endocytosis’ provided a description of the current state of the art of this exciting and dynamic field and identified major challenges for the years to come. The meeting underscored the fact that a comprehensive understanding of the biology of the endolysosomal system spanning from the detailed mechanistic understanding of individual endocytic events to the analysis of integrated system properties in complex multicellular environments will require the use of interdisciplinary approaches that range from state-of-the-art cell biology and biochemistry to quantitative biophysics, theoretical modeling, systems biology and in vivo studies. Traffic 2014; 15: 338–346
Acknowledgments We thank Ludger Johannes and Marcos González-Gaitán for their role in the organization of the meeting and various colleagues for their comments on the manuscript. We apologize to meeting participants whose contributions we have not been able to include due to space constraints. We are particularly grateful to Tom Kirchhausen and Marko Kaksonen for providing illustrations for this review.
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14. Romer W, Pontani LL, Sorre B, Rentero C, Berland L, Chambon V, Lamaze C, Bassereau P, Sykes C, Gaus K, Johannes L. Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell 2010;140:540–553. 15. Abrami L, Brandi L, Moayeri M, Brown MJ, Krantz BA, Leppla SH, van der Goot FG. Hijacking multivesicular bodies enables long-term and exosome-mediated long-distance action of anthrax toxin. Cell Rep 2013;5:986–996. 16. Parton RG, del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol 2013;14:98–112. 17. Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, Stan RV, Butler-Browne G, Vedie B, Johannes L, Morone N, Parton RG, Raposo G, Sens P, Lamaze C, et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 2011;144:402–413. 18. Ludwig A, Howard G, Mendoza-Topaz C, Deerinck T, Mackey M, Sandin S, Ellisman MH, Nichols BJ. Molecular composition and ultrastructure of the caveolar coat complex. PLoS Biol 2013;11:e1001640. 19. Hansen CG, Shvets E, Howard G, Riento K, Nichols BJ. Deletion of cavin genes reveals tissue-specific mechanisms for morphogenesis of endothelial caveolae. Nat Commun 2013;4:1831. 20. Blouin CM, Lamaze C. Interferon gamma receptor: the beginning of the journey. Front Immunol 2013;4:267. 21. Henne WM, Buchkovich NJ, Zhao Y, Emr SD. The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices. Cell 2012;151:356–371. 22. Buchkovich NJ, Henne WM, Tang S, Emr SD. Essential N-terminal insertion motif anchors the ESCRT-III filament during MVB vesicle formation. Dev Cell 2013;27:201–214. 23. Bissig C, Gruenberg J. Lipid sorting and multivesicular endosome biogenesis. Cold Spring Harb Perspect Biol 2013;5:a016816. 24. Kavaliauskiene S, Nymark CM, Bergan J, Simm R, Sylvanne T, Simolin H, Ekroos K, Skotland T, Sandvig K. Cell density-induced changes in lipid composition and intracellular trafficking. Cell Mol Life Sci 2013; (In press) doi: 10.1007/s00018-013-1441-y. 25. Kozik P, Hodson NA, Sahlender DA, Simecek N, Soromani C, Wu J, Collinson LM, Robinson MS. A human genome-wide screen for regulators of clathrin coated vesicle formation reveals an unexpected role for the V-ATPase. Nat Cell Biol 2013;15:50–60. 26. Kay JG, Koivusalo M, Ma X, Wohland T, Grinstein S. Phosphatidylserine dynamics in cellular membranes. Mol Biol Cell 2012;23:2198–2212. 27. Poteryaev D, Datta S, Ackema K, Zerial M, Spang A. Identification of the switch in early-to-late endosome transition. Cell 2010;141:497–508.
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28. Giordano F, Saheki Y, Idevall-Hagren O, Colombo SF, Pirruccello M, Milosevic I, Gracheva EO, Bagriantsev SN, Borgese N, De Camilli P. PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 2013;153:1494–1509. 29. Eden ER, White IJ, Tsapara A, Futter CE. Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction. Nat Cell Biol 2010;12:267–272. 30. Michaillat L, Baars TL, Mayer A. Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1. Mol Biol Cell 2012;23:881–895. 31. Collinet C, Stoter M, Bradshaw CR, Samusik N, Rink JC, Kenski D, Habermann B, Buchholz F, Henschel R, Mueller MS, Nagel WE, Fava E, Kalaidzidis Y, Zerial M. Systems survey of endocytosis by multiparametric image analysis. Nature 2010;464:243–249. 32. Sigismund S, Algisi V, Nappo G, Conte A, Pascolutti R, Cuomo A, Bonaldi T, Argenzio E, Verhoef LG, Maspero E, Bianchi F, Capuani F, Ciliberto A, Polo S, Di Fiore PP. Threshold-controlled ubiquitination of the EGFR directs receptor fate. EMBO J 2013;32:2140–2157. 33. Medina DL, Fraldi A, Bouche V, Annunziata F, Mansueto G, Spampanato C, Puri C, Pignata A, Martina JA, Sardiello M, Palmieri M, Polishchuk R, Puertollano R, Ballabio A. Transcriptional activation of lysosomal exocytosis promotes cellular clearance. Dev Cell 2011;21:421–430. 34. Settembre C, De Cegli R, Mansueto G, Saha PK, Vetrini F, Visvikis O, Huynh T, Carissimo A, Palmer D, Klisch TJ, Wollenberg AC, Di Bernardo D, Chan L, Irazoqui JE, Ballabio A. TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop. Nat Cell Biol 2013;15:647–658. 35. Battich N, Stoeger T, Pelkmans L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat Methods 2013;10:1127–1133. 36. Coumailleau F, Fürthauer M, Knoblich JA, Gonzalez-Gaitan M. Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division. Nature 2009;458:1051–1055. 37. de la Roche M, Ritter AT, Angus KL, Dinsmore C, Earnshaw CH, Reiter JF, Griffiths GM. Hedgehog signaling controls T cell killing at the immunological synapse. Science 2013;342:1247–1250. 38. Tanaka H, Kitakura S, Rakusova H, Uemura T, Feraru MI, De Rycke R, Robert S, Kakimoto T, Friml J. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana. PLoS Genet 2013;9:e1003540.
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Traffic Report
Systems Dynamics in Endocytosis Maximilian Fürthauer1∗ and Elizabeth Smythe2∗ 1 Institut 2 ∗
de Biologie Valrose, CNRS UMR7277, INSERM 1091, University of Nice Sophia-Antipolis, Nice, France Centre for Membrane Interactions and Dynamics, Department of Biomedical Science, University of Sheffield, Sheffield, UK
Corresponding authors: Maximilian Fürthauer, [email protected] and Elizabeth Smythe, [email protected]
Abstract The endocytic system acts at the crossroads of different cellular activities
to complement the insights that can be gained by biochemical and cell
to play a central role in the regulation of cell signaling and membrane
biological approaches with the use of quantitative biophysics, systems
dynamics. An European Molecular Biology Organization (EMBO) confer-
biology and animal model systems to achieve an integrated view of
ence held in October 2013 in Villars-sur-Ollon gathered researchers from
the properties of the endomembrane system and its role in cellular
all over the world to present their latest findings on the endolysosomal
information processing.
system and identify major challenges for the future. The conference covered the entire spectrum of research in this rapidly evolving field
Keywords endocytosis, lipids, membrane dynamics, signaling, systems biology
ranging from the cellular mechanics of endocytosis to the role of proteins and lipids in the biogenesis and function of endolysosomal
Received 10 December 2013, revised and accepted for publication 22
organelles and the analysis of higher order system properties in multi-
December 2013, uncorrected manuscript published online 9 January
cellular contexts. In particular, the meeting highlighted current efforts
2014
The year 2013 saw the passing of Christian de Duve, the discoverer of lysosomes and 1974 Nobel Prize in Physiology and Medicine winner. In 1963, de Duve coined the term ‘endocytosis’ to designate a process in which a plasma membrane invagination leads to the formation of intracellular carrier vesicles that allow the engulfment of particles or fluids from the extracellular environment (1). An EMBO conference organized by Marcos GonzálezGaitán (University of Geneva, Switzerland) and Ludger Johannes (Institut Curie, Paris, France) on the ‘Systems Dynamics of Endocytosis’ provided ample evidence that 50 years after de Duve’s naming, research in the field of endocytosis is more vibrant than ever.
the plasma membrane, the cytoplasmic organelles as well as biosynthetic and degradative compartments. The endolysosomal system has emerged as a central regulator of cellular activities that govern not only nutrient uptake and signal interpretation from the cell surface, but also the regulation of cellular metabolism and the capacity of a cell to communicate with its neighbors. It is therefore no surprise that dysfunctions of this system have been shown to be highly deleterious to animal development and linked to a number of human diseases. The conference presentations reflected this breadth.
The Cellular Mechanics of Endocytosis Five decades of research have indeed revealed that the importance of endocytic trafficking extends far beyond the mere uptake of materials from the extracellular space. The paramount importance of this system is due to its ability to serve as a cellular highway that interconnects traffic between different cellular compartments including 338 www.traffic.dk
Where it all starts: the growing diversity of endocytic entry portals Clathrin-dependent endocytosis While clathrin-mediated endocytosis (CME) is undoubtedly the most studied and best understood endocytic
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entry route, a number of lively discussions highlighted the controversies that still surround this complex process. However, the discussions also revealed that a consensus is emerging with respect to key aspects of clathrin-coated pit (CCP) and vesicle (CCV) formation. Live cell imaging is unraveling the dynamics of CCP formation at the plasma membrane, demonstrating that it is a stochastic process. Clathrin assemblies can initiate and then disassemble (abort) very rapidly. Tom Kirchhausen (Harvard Medical School, Boston, MA, USA) described how CCP initiation requires the cooperative use of weak interactions between the plasma membrane phospholipid PI(4,5)P2 , the endocytic adapter protein AP2 and clathrin. Total Internal Reflection Fluorescence (TIRF) microscopy with single molecule sensitivity has determined that a stoichiometry of 2 AP2 molecules per clathrin triskelion is required for the initiation of CCP formation but not sufficient to guarantee further CCP to CCV maturation (2), (Figure 1). While early CCPs may still undergo disassembly, Kirchhausen proposed that gatekeepers such as FCHo proteins and cargo stabilise the maturing pit leading to productive CCV formation (3). Sandy Schmid (UT Southwestern, Dallas, TX, USA) extended the quantitative analysis of CME to include CCP maturation, that precedes dynamin-dependent scission to form a CCV (4). Her study revealed that many pits also abort relatively late (up to 20 seconds in a 60 second time-scale), suggesting the existence of specific checkpoints, analogous to the gatekeepers discussed above, which regulate maturation. Dynamin recruitment is key to functional CCV formation and so may be one such checkpoint. Consistent with previous studies (5), dynamin was observed to be recruited early in CCP assembly. A second checkpoint involves the diverse family of endocytic accessory proteins (EAPs) which contribute to recruitment of specific cargo and curvature generation at CCPs. Analysis of an AP2 mutant that is impaired for EAP binding revealed a surprising robustness of CME: while a failure in EAP recruitment resulted in an increased frequency of abortive CCP assembly events, this defect was compensated for by an increase in the overall number of initiation events and the speed of CCP/CCV maturation, thus explaining why endocytosis of housekeeping cargo such as transferrin receptor is unaffected in the absence Traffic 2014; 15: 338–346
Figure 1: Clathrin coated pit assembly. The cartoon shows how two AP2 molecules tether a single clathrin triskelion to the inner surface of the plasma membrane in the initial stages of clathrin lattice assembly. As more AP2 complexes and clathrin assemble, there is increased membrane curvature until scission results in a coated vesicle. Courtesy of Tom Kirchhausen, Harvard University. of EAP binding (6). The development of sensitive assays to monitor CCP dynamics in the context of individual pits containing specific cargo will greatly facilitate our understanding of the roles of EAPs. Expanding on the theme of cargo determining coated pit dynamics, Liz Smythe (University of Sheffield, UK) described how clathrin light chain phosphorylation helps to define subpopulations of CCPs containing different G protein-coupled receptors and how this segregation may impact on downstream signaling (7). Both Kirchhausen and Schmid emphasized the methodological challenges associated with quantitative live cell imaging and the necessity to have unambiguous definitions, e.g. the criteria used to define productive versus 339
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abortive CME events. There was a discussion of the relative merits of overexpression of endocytic proteins versus genome-edited cells that express functional green fluorescent protein (GFP)-fusion proteins at endogenous levels which has been heralded as the gold standard for the future (8). This would apply especially to EAPs which compete with each other for common AP2 binding sites and should therefore not be overexpressed. Sandy Schmid highlighted that the intrinsically low fluorescence obtained in genome-edited cells may prevent the proper tracking and interpretation of labeled structures. For example, previous claims that overexpression of tagged clathrin light chains impairs CME (8) are likely to reflect inaccuracies in CCP tracking due to low signal-to-noise ratios. Having a fiduciary marker of high signal-to-noise ratio substantially improves data analysis and interpretation (4). A key element to facilitate understanding of the complexity of the endocytic system from the molecular through to the organismal level will be the availability of well-characterized toolboxes to systematically investigate protein function. Tom Kirchhausen initiated a discussion on the potential to establish a resource of genome-edited mammalian cells expressing key components of the endocytic pathway which would then provide a valuable and robust experimental system for the entire community. Aur´elien Roux (University of Geneva, Switzerland) presented a radically different approach to understanding how the biophysical properties of membranes contribute to membrane scission. His studies used purified components to reconstitute individual endocytic events in vitro to reveal that membrane scission depends on membrane tension and bending rigidity as well as on the torque of the constricting dynamin helix (9). A local change in membrane curvature at the edge of the dynamin helix results in a local variation in line tension and membrane elastic energy that determines the location of the scission event. Extending this type of approach to the analysis of CME can generate a biophysical phase diagram that describes the transition from an initial flat sheet of membrane, through a shallow CCP and toward the mature CCV depending on the local biophysical properties of the system. The importance of the biophysical properties of the membrane is supported by differing requirements for CME depending on context. In this regard a consensus 340
is emerging that actin, the involvement of which in mammalian endocytosis has long been debated, is required in environments with elevated membrane tension (10) such as the rigid cholesterol/sphingolipid-rich apical surface of epithelial cells as opposed to the more floppy basolateral membrane. Clathrin links to actin via interactions of clathrin light chains with Hip1 and Hip1R, actin binding proteins. Frances Brodsky (UCSF, USA) discussed how clathrin function is surprisingly diverse and extends beyond classical CCV formation (11). One example of this is clathrin-dependent recruitment of actin which facilitates internalization of bacteria via the clathrin light chain-Hip pathway (12). A challenge for the future is imaging of clathrin dynamics in vivo in vertebrate systems. Tom Kirchausen showed some intriguing imaging using both whole cell live cell imaging and intra vital microscopy indicating that such approaches are not far off.
Clathrin-independent endocytosis In addition to CME, cells use a wide variety of alternative, clathrin-independent endocytosis (CIE) pathways, whose inter-relationships and regulation is similarly complex. Nathalie Sauvonnet (Institut Pasteur, Paris, France) presented novel insights into the trafficking of the Interleukin 2 cytokine Receptor (IL-2R) which is internalized via a CIE pathway that requires Dynamin, Phosphatidylinositol-3-Kinase and an actin-regulator NWASP (13). Ultrastructural analysis of cells undergoing IL-2R endocytosis suggests that this process may involve protrusions emerging from the cell surface. Synaptic vesicle recycling endocytosis has to occur on a much faster time-scale (within a few seconds) than classical CME. Harvey McMahon (MRC-LMB, Cambridge, UK) presented evidence for the existence of a novel CIE pathway that might fulfill these kinetic requirements and which, while active in non-neuronal cells, is likely to be essential in neurons. A major technical issue for the future will be to achieve a direct visualization of fast endocytic events in the physiological context of the neuronal synapse. As for CME, pathways for CIE have been uncovered through the analysis of pathogenic toxins that induce their own endocytosis. Ludger Johannes described the Traffic 2014; 15: 338–346
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capacity of the B-Subunit of Shiga Toxin to bind the glycosphingolipid Gb3 at the cell surface, generate membrane curvature and trigger actin-dependent scission of endocytic carrier vesicles (14). For the Shiga toxin uptake pathway, his group has now identified a BAR domain protein that is required not for initial membrane bending (that is driven by the toxin), but for subsequent scission in synergy with actin. Other recent data suggest that glycosphingolipids also play a role in physiological, non-pathogenic endocytosis of endogenous cargo proteins such as CD44, and that this process is driven by endogenous lectins by mechanisms similar to the one discovered for Shiga toxin. While self-induced endocytosis of bacterial toxins is an essential first step for entry into host cells, toxins must ultimately leave their endocytic vehicle to enter the cytoplasm and exert their effect on host cell physiology. Gisou van der Goot (EPFL, Lausanne, Switzerland) presented the case of the anthrax toxin lethal factor (LF) which takes advantage of the pore-forming protective antigen to translocate into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs). Back-fusion of ILVs with the endosomal limiting membrane is then proposed to release LF into the cytoplasm. New data reveal that MVEs do not only ensure immediate LF release into the cytoplasm but that they can also act as long term toxin reservoirs, the content of which can be released by exosomes into the extracellular medium even after the bacteria themselves have been cleared from the organism (15). Intriguingly, this may indicate either the existence of a subpopulation of stable late endosomal compartments or the presence of both short and long lived ILVs within a common MVE. Membrane microdomains: from endocytosis to stress response and cell signaling Rob Parton (IMB, Brisbane, Australia) illustrated that internalization of plasma membrane components is not always linked to cargo uptake. While endocytosis of caveolae can account for a substantial fraction of total cellular endocytic uptake under certain circumstances, it is becoming evident that the primary function of caveolar endocytosis is to regulate their own abundance at the cell surface (16). In this context cells can respond to mechanical stress by caveolar disassembly as a protective mechanism to ensure membrane integrity (17), an Traffic 2014; 15: 338–346
observation that may shed novel light on muscular dystrophies resulting from human caveolar dysfunction. Interestingly membrane stretching disrupts the interactions between caveolins and their essential cavin partner proteins. As cavins have initially been described as nuclear proteins this begs the question whether the membrane release and nuclear translocation of these molecules may trigger stress-induced signaling. By combining biochemical analyses, mass spectrometry and high resolution electron microscopy Benjamin Nichols (MRC-LMB, Cambridge, UK) provided evidence that caveolae are composed of repeated units of a caveolar coat complex composed of cavins and caveolins (18). Knockout mice for cavin2 and cavin3 reveal that despite this unifying architecture, deletion of individual cavin genes results in tissue-specific phenotypes that are indicative of caveolar heterogeneity (19). While the main role of caveolae appears to be as a structural membrane organizer, membrane microdomains have long been proposed to play an essential role in the dynamic assembly of cellular signaling platforms. Experimental support to substantiate this attractive hypothesis has however been difficult to obtain. Christophe Lamaze (Institut Curie, Paris, France) presented evidence for an essential role of plasma membrane rafts in the regulation of cell signaling by the Interferon Gamma receptor (20). Structural analysis of the endocytic machinery While live imaging and functional assays have yielded a wealth of information with respect to the roles and hierarchy of individual endocytic proteins, a major challenge is to determine at the molecular level how multiprotein machines assemble. Two studies illustrated the use of latest-generation electron microscopy to bridge the gap between the structure and the function of the endocytic machinery. Marko Kaksonen (EMBL, Heidelberg, Germany) presented an elegant combination of correlative electron tomography (20) and single particle tracking to determine the dynamics of recruitment of endocytic proteins during CME in yeast. The power of this approach is highlighted by its capacity to measure the number of molecules involved in a given event as well as providing information about the 341
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100 nm
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Figure 2: Correlative electron microscopy is a powerful tool to understand how endocytic proteins contribute to CCP structure. The left panel shows a centroid trajectory of a GFP-tagged yeast endocytic coat protein patch during vesicle budding. The right panel shows an endocytic membrane invagination identified by fluorescence microscopy and then imaged at high resolution by electron tomography. Courtesy of Andrea Picco, Wanda Kukulsk and Marko Kaksonen (EMBL). spatial orientation of a molecule during macromolecular assembly (Figure 2). Using in vitro assembly of purified protein components on defined lipid templates, Scott Emr (Cornell University, Ithaca, NY, USA) provided structural information about the assembly of the ESCRTIII machinery that forms membrane-shaping helices required for membrane scission during the biogenesis of ILVs of MVEs, cytokinetic abscission and viral budding. While oligomerization of the major ESCRTIII component SNF7/CHMP4 is sufficient for the formation of flat spirals on lipid monolayers, incorporation of Vps24 and Vps2 triggers the transformation of these 2-dimensional spirals into 3dimensional helices that may act to deform associated membranes (21). Combined assembly of ESCRTII and III subcomplexes leads to the formation of a macromolecular ring that may confine membrane and the cargo content of a single ILV (22).
Biogenesis, Function and Connectivity of Endolysosomal Organelles Lipids end endocytosis While technical limitations have for a long time confined the vast majority of cell biological studies to the analysis of protein components, it is becoming clear that a comprehensive understanding of the endolysosomal 342
system will depend on our ability to characterize the functional contribution of the many lipid species that compose different cellular membranes. Cellular cholesterol is mostly derived from the endocytic uptake of low density lipoproteins and their subsequent de-esterification and mobilization from late endosomal compartments. Jean Gruenberg (University of Geneva, Switzerland) illustrated the importance of the late endosomal phospholipid lysobisphosphatidic acid (LBPA) for the export of endocytosed cholesterol through a poorly understood trafficking route that may involve the back-fusion of ILVs with the late endosomal limiting membrane, a process that may be mechanistically related to the endosomal egress route used by Vesicular Stomatitis Virus (23). In support of this hypothesis, functional screens for the regulation of endosomal lipid composition have identified cellular factors that are required for both processes. The impact of lipid composition on endosomal trafficking was illustrated by Kirsten Sandvig (University of Oslo, Norway). Lipidomic analysis of cells grown at various densities reveals major changes in different lipid species (24) which correlated with significant changes in the cell surface binding and retrograde transport of Shiga toxin. Different cell types differ in their density-dependent regulation of Shiga toxin receptor levels but not for the density-dependence of Shiga toxin binding to the cell surface. These observations suggest that toxin/receptor Traffic 2014; 15: 338–346
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binding is not governed by abundance only but also by the lipid environment of receptor molecules. In the course of an RNAi-based screen designed to identify novel regulators of CME, Margaret Robinson (CIMR, Cambridge, UK) discovered an unexpected link between the vacuolar V-ATPase and the regulation of plasma membrane cholesterol (25). Ultrastructural analysis revealed the presence of non-constricted clathrin coated invaginations at the surface of V-ATPase depleted cells. The observation that the appearance of these aberrant structures can be rescued by cholesterol addition suggests that V-ATPase activity may be required for the transport of cholesterol from endosomes to the plasma membrane. Sergio Grinstein (Hospital for Sick Children, Toronto, Canada) illustrated the use of a fluorescent probe based on the lactadherin C2 domain to follow the cellular dynamics of phosphatidylserine (PS), a major component of the inner plasma membrane leaflet (26). A functional screen of nonessential genes in the yeast genome led to the discovery that ergosterol (the yeast equivalent of cholesterol) is important for PS distribution. This effect on PS can be explained by the importance of cholesterol for endosomal recycling, highlighting the fact that endocytic trafficking is not only lipid-dependent but also essential to maintain the cellular distribution of the lipids themselves. Biogenesis and functional connections of endolysosomal organelles Anne Spang (Biozentrum, Basel, Switzerland) described mechanisms by which one organelle can be converted to another by studying the conversion of Rab5-positive early endosomes into Rab7-positive late endosomes (27) and the role of HOPS and Corvet complexes. The macrophagelike coelomocytes of the nematode Caenorhabditis elegans present a natural population of gigantic endosomes that allow visualization of endosomal maturation processes using light microscopy. A major question for future studies will be to determine whether endosome conversion is regulated by the biophysical properties of the system (e.g. phosphoinositide composition) or the direct monitoring of organelle size. Two presentations illustrated how different cellular organelles can communicate through the use of specialized Traffic 2014; 15: 338–346
contact sites. Using a combination of imaging approaches from electron microscopy to optogenetic manipulation of cellular phosphoinositide levels, Francesca Giordano (De Camilli Lab, Yale School of Medicine, New Haven, CT, USA) provided evidence for the existence of contact sites, mediated by extended synaptotagmins, between the endoplasmic reticulum (ER) and the plasma membrane, that are functionally distinct from previously characterized contact sites regulated by STIM1 and Ora1 (28). While functional connections between the endosomal system and the Golgi apparatus have long been known, it has only recently become evident that specialized contact zones also exist between endosomes and the ER (29). Harald Stenmark (University of Oslo, Norway) presented evidence for the importance of ER-endosome contact sites in the regulation of endosome motility as vesicles switch from an initial centripetal to a later centrifugal movement through the recruitment of specific motor proteins. Cells modulate the size and composition of their organelles in response to environmental conditions. Andreas Mayer (University of Lausanne, Switzerland) raised the question of whether the content of an organelle influences its size? The size of the yeast vacuole is regulated in response to nutrient availability (30). A single molecular species, inorganic poly-phosphate accounts for the vast majority of vacuolar content. Consequently manipulations of polyphosphate synthesis induce significant alterations in vacuolar content and impact on organelle size and number. It will be of interest to know the relative contributions of intrinsic physicochemical properties and extrinsic sensing mechanisms in the control of organelle size.
Integrative Approaches for the Analysis of the Endolysosomal System Systems biology of endocytosis Beyond the mechanistic characterization of individual endocytic events, a number of presentations highlighted current efforts to achieve an understanding of endomembrane system using systems biology approaches. Marino Zerial (MPI-CBG, Dresden, Germany) presented studies on the higher order properties of the endocytic system with respect to the regulation of cellular signal transduction (31). Through a ground-breaking quantitative 343
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analysis of endosomal signal transduction he provided evidence that the endocytic system acts as a cellular information processing centre that converts incoming external stimuli into digitalized biological information. Interestingly, differences in this cellular information processing can account for the capacity of different growth factors to generate different biological responses while sharing common downstream components. Pier Paolo Di Fiore (IFOM, Milan, Italy) raised the question of how the wiring of the endocytic system enables it to accommodate the wide range of EGF concentrations that are encountered in vivo. Low EGF concentrations induce CME and receptor recycling, while high concentrations induce ubiquitination-dependent CIE, and subsequent receptor degradation (32). The threshold-controlled ubiquitination that is required for CIE converts a gradient of increasing EGF concentration into a threshold-activated ON/OFF response. A mathematical model of EGF/EGFR endocytosis describes how this behavior can account for the capacity of the system to cope with a wide range of ligand concentrations which can differ over two orders of magnitude in normal physiological conditions. Strikingly, the model reveals that system robustness is observed only within certain limits and that these may be surpassed by the extremely high EGF concentrations present in cancerous tissues.
of cell–cell interactions. Lukas Pelkmans (University of Zürich, Switzerland) approached the influence of cell density on the properties of the endocytic system. The use of a newly established approach for imagebased transcriptomics (35) combines information about the crowding status of individual cells with their transcriptional response. The use of this methodology permits the identification of the factors involved in the sensing of cell crowding as well as the downstream mediators of the cellular crowding response. It is particularly striking to note that this type of analysis reveals how social behavior of cells can be generated in a purely cell-intrinsic way, without the necessity for dedicated pathways of cell-to-cell communication. From cells to tissues: endocytic trafficking in multicellular organisms One of the most obvious characteristics of multicellular organisms is the organization of cells into polarized tissues of defined spatial orientation. Four presentations dealt with the impact of cell and tissue polarity on the regulation of the cellular endomembrane system. Through a large-scale analysis of key regulators of cellular membrane trafficking in different fly tissues Susanne Eaton (MPI-CBG, Dresden, Germany) revealed that there is no universal relationship between cell polarity and the positioning of endolysosomal organelles in polarized tissues. Rather than being stereotypes, the positioning of organelles appears to be highly variable and adapted to the specific requirements of a given cellular context.
Following de Duve’s pioneering discovery of lysosomes it has become clear that this organelle is more than simply a cellular garbage disposal unit since it also plays essential roles in homeostasis through the regulation of protein degradation, autophagy and lysosomal exocytosis. Lysosomal adaptation posits that the entire spectrum of functions of an organelle is adjusted to the demands of cellular physiology by a central master regulator. Taking advantage of a systems biology approach, Andrea Ballabio (TIGEM, Naples, Italy) presented the transcription factor TFEB as a master regulator of lysosomal activities. The potency of this master regulator is further illustrated by the striking observation that the overexpression of TFEB rescues lysosomal storage disorders (33) and enhances lipid degradation in mouse models of obesity (34).
Accordingly, Emmanuel Derivery from the lab of Marcos González-Gaitán (University of Geneva, Switzerland) described the mechanisms through which the polarization of an asymmetrically dividing Drosophila sensory precursor cells drives the directional movement of a population of signaling endosomes that can be identified through the presence of the endosomal adapter protein SARA and ensure the transport of Delta ligands and their cognate Notch receptors (36). Careful quantitative analysis of individual endosome motility in high resolution time lapse recording reveals the role of the polarized cytoskeleton in the regulation of asymmetric endosome segregation.
One of the fundamental characteristics of living organisms is the variability of their constituent cells and the roles
Gillian Griffiths (CIMR, Cambridge, UK) described the regulation of polarized exocytosis at the immune synapse.
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In this context cellular polarity is not governed by the overall organization of the tissue but rather by the site of interaction between cytotoxic T-cells and antigenpresenting cells. Following activation, T-cells promote the biogenesis of secretory lysosomes. The polarized delivery of these cytotoxic organelles is determined by the directional movement of the T-cell centrosome toward the immunological synapse (the specialized contact zone between a T-cell and antigen-presenting cell) and polarization of the cytoskeleton. Most interestingly, a thorough cell biological analysis of activated T-cell behavior suggests that the immunological synapse may share important properties with primary cilia, including a role for hedgehog (Hh) signaling in T lymphocytes. However, in contrast to Hh signaling in the cilium, Hh signaling is mediated by an intracellular pool of Indian Hh which colocalizes with the its receptor (Patched) on a subset of endosomes (37). Jiˇrí Friml (ISTA, Klosterneuburg, Austria) illustrated the importance of polarized endocytic trafficking for the generation of directional gradients of the plant hormone Auxin that play a major role in plant morphogenesis. Directional Auxin transport depends on the polarized localization of Pin-formed (PIN) auxin transporters that can be regulated by intrinsic developmental and extrinsic environmental cues (38). PIN localization is regulated through continuous CME and endosomal recycling that ensures rapid shuttling of molecules between endosomes and the different polar domains at the plasma membrane. A computer model that integrates the dynamics of PIN trafficking with feedback regulation by auxin can fully account for the role of this major plant hormone in plant embryonic development. Altogether, the EMBO conference on ‘Systems Dynamics of Endocytosis’ provided a description of the current state of the art of this exciting and dynamic field and identified major challenges for the years to come. The meeting underscored the fact that a comprehensive understanding of the biology of the endolysosomal system spanning from the detailed mechanistic understanding of individual endocytic events to the analysis of integrated system properties in complex multicellular environments will require the use of interdisciplinary approaches that range from state-of-the-art cell biology and biochemistry to quantitative biophysics, theoretical modeling, systems biology and in vivo studies. Traffic 2014; 15: 338–346
Acknowledgments We thank Ludger Johannes and Marcos González-Gaitán for their role in the organization of the meeting and various colleagues for their comments on the manuscript. We apologize to meeting participants whose contributions we have not been able to include due to space constraints. We are particularly grateful to Tom Kirchhausen and Marko Kaksonen for providing illustrations for this review.
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