Endosomal sorting sorted - motors, adaptors and lessons from in vitro and cellular studies.

Motor proteins are key players in exerting spatiotemporal control over the intracellular location of membrane-bound compartments, including endosomes containing cargo. In this Review, we focus on how motors and their cargo adaptors regulate positioning of cargoes from the earliest stages of endocytosis and through the two main intracellular itineraries: (1) degradation at the lysosome or (2) recycling back to the plasma membrane. In vitro and cellular (in vivo) studies on cargo transport thus far have typically focussed independently on either the motor proteins and adaptors, or membrane trafficking. Here, we will discuss recent studies to highlight what is known about the regulation of endosomal vesicle positioning and transport by motors and cargo adaptors. We also emphasise that in vitro and cellular studies are often performed at different scales, from single molecules to whole organelles, with the aim to provide a perspective on the unified principles of motor-driven cargo trafficking in living cells that can be learned from these differing scales.

Plasminogen Receptors Promote Lipoprotein(a) Uptake by Enhancing Surface Binding and Facilitating Macropinocytosis.

BACKGROUND: High levels of Lp(a) (lipoprotein(a)) are associated with multiple forms of cardiovascular disease. Lp(a) consists of an apoB100-containing particle attached to the plasminogen homologue apo(a). The pathways for Lp(a) clearance are not well understood. We previously discovered that the plasminogen receptor PlgRKT (plasminogen receptor with a C-terminal lysine) promoted Lp(a) uptake in liver cells. Here, we aimed to further define the role of PlgRKT and to investigate the role of 2 other plasminogen receptors, annexin A2 and S100A10 (S100 calcium-binding protein A10) in the endocytosis of Lp(a). METHODS: Human hepatocellular carcinoma (HepG2) cells and haploid human fibroblast-like (HAP1) cells were used for overexpression and knockout of plasminogen receptors. The uptake of Lp(a), LDL (low-density lipoprotein), apo(a), and endocytic cargos was visualized and quantified by confocal microscopy and Western blotting. RESULTS: The uptake of both Lp(a) and apo(a), but not LDL, was significantly increased in HepG2 and HAP1 cells overexpressing PlgRKT, annexin A2, or S100A10. Conversely, Lp(a) and apo(a), but not LDL, uptake was significantly reduced in HAP1 cells in which PlgRKT and S100A10 were knocked out. Surface binding studies in HepG2 cells showed that overexpression of PlgRKT, but not annexin A2 or S100A10, increased Lp(a) and apo(a) plasma membrane binding. Annexin A2 and S100A10, on the other hand, appeared to regulate macropinocytosis with both proteins significantly increasing the uptake of the macropinocytosis marker dextran when overexpressed in HepG2 and HAP1 cells and knockout of S100A10 significantly reducing dextran uptake. Bringing these observations together, we tested the effect of a PI3K (phosphoinositide-3-kinase) inhibitor, known to inhibit macropinocytosis, on Lp(a) uptake. Results showed a concentration-dependent reduction confirming that Lp(a) uptake was indeed mediated by macropinocytosis. CONCLUSIONS: These findings uncover a novel pathway for Lp(a) endocytosis involving multiple plasminogen receptors that enhance surface binding and stimulate macropinocytosis of Lp(a). Although the findings were produced in cell culture models that have limitations, they could have clinical relevance since drugs that inhibit macropinocytosis are in clinical use, that is, the PI3K inhibitors for cancer therapy and some antidepressant compounds.

Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation.

BACKGROUND: T cell activation leads to increased expression of the receptor for the iron transporter transferrin (TfR) to provide iron required for the cell differentiation and clonal expansion that takes place during the days after encounter with a cognate antigen. However, T cells mobilise TfR to their surface within minutes after activation, although the reason and mechanism driving this process remain unclear. RESULTS: Here we show that T cells transiently increase endocytic uptake and recycling of TfR upon activation, thereby boosting their capacity to import iron. We demonstrate that increased TfR recycling is powered by a fast endocytic sorting pathway relying on the membrane proteins flotillins, Rab5- and Rab11a-positive endosomes. Our data further reveal that iron import is required for a non-canonical signalling pathway involving the kinases Zap70 and PAK, which controls adhesion of the integrin LFA-1 and eventually leads to conjugation with antigen-presenting cells. CONCLUSIONS: Altogether, our data suggest that T cells boost their iron importing capacity immediately upon activation to promote adhesion to antigen-presenting cells.

Recycling of Apolipoprotein(a) After PlgRKT-Mediated Endocytosis of Lipoprotein(a).

RATIONALE: Lipoprotein(a) [Lp(a)] is a low-density lipoprotein-like lipoprotein and important cardiovascular risk factor whose cognate receptor and intracellular fate remains unknown. OBJECTIVE: Our study aimed to determine the intracellular trafficking pathway for Lp(a) and the receptor responsible for its uptake in liver cells. METHODS AND RESULTS: Human hepatoma cells were treated with Lp(a) purified from human plasma and Lp(a) uptake studied using Western blot analysis and intracellular localization of Lp(a) by confocal microscopy. Lp(a) was maximally internalized by 2 hours and was detected by an antiapo(a) antibody to be localized to Rab5-positive early endosomes, the trans-Golgi network, and subsequently Rab11-positive recycling endosomes. In human hepatoma cells, the apo(a) component from the internalized Lp(a) was resecreted back into the cellular media, whereas the low-density lipoprotein component was localized to the lysosomal compartment. Lp(a) internalization was reduced 0.35-fold in HAP1 and 0.33-fold in human hepatoma cells in which the plasminogen receptor (KT) was knocked out. Conversely, Lp(a) internalization was enhanced 2-fold in HAP1 and 1.6-fold in human hepatoma cells in which plasminogen receptor (KT) was overexpressed, showing for the first time the role of a specific plasminogen receptor in Lp(a) uptake. CONCLUSIONS: The novel findings that Lp(a) is internalized by the plasminogen receptor, plasminogen receptor (KT), and the apo(a) component is recycled may have important implications for the catabolism and function of Lp(a).

Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins.

Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.

Calpains, cleaved mini-dysferlinC72, and L-type channels underpin calcium-dependent muscle membrane repair.

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Serotonin: an overlooked regulator of endocytosis and endosomal sorting?

Serotonin is a neurotransmitter and a hormone that is typically associated with regulating our mood. However, the serotonin transporter and receptors are expressed throughout the body, highlighting the much broader, systemic role of serotonin in regulating human physiology. A substantial body of data strongly implicates serotonin as a fundamental regulator of endocytosis and endocytic sorting. Serotonin has the potential to enhance endocytosis through three distinct mechanisms - serotonin signalling, serotonylation and insertion into the plasma membrane - although the interplay and relationship between these mechanisms has not yet been explored. Endocytosis is central to the cellular response to the extracellular environment, controlling receptor distribution on the plasma membrane to modulate signalling, neurotransmitter release and uptake, circulating protein and lipid cargo uptake, and amino acid internalisation for cell proliferation. Uncovering the range of cellular and physiological circumstances in which serotonin regulates endocytosis is of great interest for our understanding of how serotonin regulates mood, and also the fundamental understanding of endocytosis and its regulation throughout the body. This article has an associated Future Leader to Watch interview with the first author of the paper.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.

Serotonin Receptor and Transporter Endocytosis Is an Important Factor in the Cellular Basis of Depression and Anxiety.

Depression and anxiety are common, debilitating psychiatric conditions affecting millions of people throughout the world. Current treatments revolve around selective serotonin reuptake inhibitors (SSRIs), yet these drugs are only moderately effective at relieving depression. Moreover, up to 30% of sufferers are SSRI non-responders. Endocytosis, the process by which plasma membrane and extracellular constituents are internalized into the cell, plays a central role in the regulation of serotonin (5-hydroxytryptophan, 5-HT) signaling, SSRI function and depression and anxiety pathogenesis. Despite their therapeutic potential, surprisingly little is known about the endocytosis of the serotonin receptors (5-HT receptors) or the serotonin transporter (SERT). A subset of 5-HT receptors are endocytosed by clathrin-mediated endocytosis following serotonin binding, while for the majority of 5-HT receptors the endocytic regulation is not known. SERT internalizes serotonin from the extracellular space into the cell to limit the availability of serotonin for receptor binding and signaling. Endocytosis of SERT reduces serotonin uptake, facilitating serotonin signaling. SSRIs predominantly inhibit SERT, preventing serotonin uptake to enhance 5-HT receptor signaling, while hallucinogenic compounds directly activate specific 5-HT receptors, altering their interaction with endocytic adaptor proteins to induce alternate signaling outcomes. Further, multiple polymorphisms and transcriptional/proteomic alterations have been linked to depression, anxiety, and SSRI non-response. In this review, we detail the endocytic regulation of 5-HT receptors and SERT and outline how SSRIs and hallucinogenic compounds modulate serotonin signaling through endocytosis. Finally, we will examine the deregulated proteomes in depression and anxiety and link these with 5-HT receptor and SERT endocytosis. Ultimately, in attempting to integrate the current studies on the cellular biology of depression and anxiety, we propose that endocytosis is an important factor in the cellular basis of depression and anxiety. We will highlight how a thorough understanding 5-HT receptor and SERT endocytosis is integral to understanding the biological basis of depression and anxiety, and to facilitate the development of a next generation of specific, efficacious antidepressant treatments.

Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

Endocytic trafficking controls the density of molecules at the plasma membrane and by doing so, the cell surface profile, which in turn determines how cells interact with their environment. A full apprehension of any cellular process necessitates understanding how proteins associated with the plasma membrane are endocytosed, how they are sorted after internalization, and if and how they are recycled to the plasma membrane. To date, it is still difficult to experimentally gain access to this information, even more to do it in a quantitative way. Here we present a toolset based on photoactivation of fluorescent proteins that enabled us to generate quantitative information on endocytosis, incorporation into sorting and recycling endosomes, delivery from endosomes to the plasma membrane, and on the type of vesicles performing intracellular transport. We illustrate these approaches by revealing striking differences in the endocytic trafficking of T-cell receptor and CD4, which bind to the same molecule at the surface of antigen-presenting cells during T-cell activation.

Membrane Heterogeneity Controls Cellular Endocytic Trafficking.

Endocytic trafficking relies on highly localized events in cell membranes. Endocytosis involves the gathering of protein (cargo/receptor) at distinct plasma membrane locations defined by specific lipid and protein compositions. Simultaneously, the molecular machinery that drives invagination and eventually scission of the endocytic vesicle assembles at the very same place on the inner leaflet of the membrane. It is membrane heterogeneity - the existence of specific lipid and protein domains in localized regions of membranes - that creates the distinct molecular identity required for an endocytic event to occur precisely when and where it is required rather than at some random location within the plasma membrane. Accumulating evidence leads us to believe that the trafficking fate of internalized proteins is sealed following endocytosis, as this distinct membrane identity is preserved through the endocytic pathway, upon fusion of endocytic vesicles with early and sorting endosomes. In fact, just like at the plasma membrane, multiple domains coexist at the surface of these endosomes, regulating local membrane tubulation, fission and sorting to recycling pathways or to the trans-Golgi network via late endosomes. From here, membrane heterogeneity ensures that fusion events between intracellular vesicles and larger compartments are spatially regulated to promote the transport of cargoes to their intracellular destination.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.

A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T cell activation.

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.

Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DE domain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.

Alternate splicing of dysferlin C2A confers Ca²âº-dependent and Ca²âº-independent binding for membrane repair.

Dysferlin plays a critical role in the Ca²âº-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²âº and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1). Interestingly, these isoforms have markedly different responses to Ca²âº and phospholipid. Structural and thermodynamic analyses are consistent with the canonical C2A domain as a Ca²âº-dependent, phospholipid-binding domain, whereas C2Av1 would likely be Ca²âº-independent under physiological conditions. Additionally, both isoforms display remarkably low free energies of stability, indicative of a highly flexible structure. The inverted ligand preference and flexibility for both C2A isoforms suggest the capability for both constitutive and Ca²âº-regulated effector interactions, an activity that would be essential in its role as a mediator of membrane repair.