Clathrin-independent endocytosis: an increasing degree of complexity

Sandvig, Kirsten; Kavaliauskiene, Simona; Skotland, Tore

doi:10.1007/s00418-018-1678-5

Cited by 163 publications

(189 citation statements)

References 110 publications

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“…Many pathways of trafficking vesicle biogenesis, including the assembly of COPII vesicles at ER exit sites and clathrin-independent endocytosis, depend upon interwoven protein networks [49][50][51] . Therefore, liquid-like catalytic complexes, which are capable of balancing the competing demands of multivalent assembly and flexible remodeling, could help to explain the efficient initiation and release of vesicles in trafficking routes throughout the cell.…”

Section: Importantly Binding Between Eps15 and Fcho1 Is Mediated Bymentioning

confidence: 99%

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Day

Kago

Wang

et al. 2019

Preprint

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During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As early initiators of endocytosis, Eps15 and Fcho1 are responsible for locally concentrating downstream components on the membrane surface. However, they must also permit dynamic rearrangement of proteins within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1 rely on weak, liquid-like interactions to efficiently catalyze endocytosis. In reconstitution experiments, these weak interactions promote the assembly of protein droplets with liquid-like properties, including rapid coalescence and dynamic exchange of protein components. To probe the physiological role of liquid-like interactions among initiator proteins, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove initiator proteins into liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15 knockout cells. In contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels.Assembly of these structures stalled vesicle budding, likely owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of early initiator proteins provides an optimal catalytic platform for endocytosis.Clathrin-mediated endocytosis (CME) is driven by the assembly of a highly interconnected network of adaptor proteins that participate in cargo selection, clathrin recruitment, and membrane bending. A small subset of adaptor proteins is the first to arrive at endocytic sites 1,2 . These initiators include the Fcho proteins (Fcho1/2), Eps15/Eps15R, and intersectin, which associate together in a complex thought to serve as a membrane-associated scaffold for recruitment of downstream adaptors 3 .Specifically, Fcho and Eps15 work together to recruit AP-2, which promotes maturation of the nascent endocytic structure 4-6 . Fcho also binds the adaptors HRB and Dab2 7,8 .Eps15 interacts with numerous other adaptors, including Epsin and AP180/CALM 9,10 .Meanwhile, the minimal initiator complex itself is highly interconnected. Fcho binds Eps15 through interactions between its C-terminal µHD and motifs in Eps15's intrinsically disordered C-terminus 6,11 . Fcho exists as a homodimer, mediated by its F-BAR domain 12 . Eps15 not only dimerizes via its coiled-coil domain, but can likely also take on an antiparallel tetrameric conformation mediated by interactions between its Nterminal EH domains and C-terminal disordered region 11,13 . Therefore, each Fcho dimer can interact with multiple Eps15 oligomers, suggesting an interconnected initiator protein network. Loss of both Fcho1 and Eps15 results in less productive CME 3,6 .As initiators of endocytosis, the network formed by Fcho and Eps15 must meet the competing demands of (i) locally concentrating downstream adaptors, while (ii) permitting them to rapidly rearrange as the endocytic structure grows and cha...

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Section: Importantly Binding Between Eps15 and Fcho1 Is Mediated Bymentioning

confidence: 99%

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Day

Kago

Wang

et al. 2019

Preprint

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“…Clathrin-mediated endocytosis (CME) is the major uptake pathway in resting cells 1,2 but additional Clathrin-independent endocytic (CIE) routes, including fast Endophilin-mediated endocytosis (FEME), perform specific functions or internalize various cargoes 3,4 . FEME is not constitutively active but is triggered upon the stimulation of selected cell surface receptors by their ligands 5 .…”

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confidence: 99%

Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

Ferreira

Casamento

Roas

et al. 2020

Preprint

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20Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Parallel to 21 Clathrin-mediated endocytosis, additional Clathrin-independent endocytic routes exist, including fast 22 Endophilin-mediated endocytosis (FEME). The latter is not constitutively active but requires the 23 activation of selected receptors. In cell culture, however, the high levels of growth factors in the 24 regular culture media induce spontaneous FEME, which can be suppressed upon serum starvation. 25Thus, we predicted a role for protein kinases in this growth factor receptor-mediated regulation of 26 the pathway. Using chemical and genetic inhibition, we found that Cdk5 and GSK3β are negative 27 regulators of FEME. Their inhibition was sufficient to activate FEME promptly in resting cells and 28 boosted the production of endocytic carriers containing β1-adrenergic receptor, following 29 dobutamine addition. We established that the kinases suppress FEME at several levels. They control 30 Dynamin-1 and Dynein recruitment and sorting of cargo receptors such as Plexin A1 and ROBO1 into 31 FEME carriers. They do so by antagonizing the binding of Endophilin to Dynamin-1 as well as to 32 Collapsin response mediator protein 4 (CRMP4), a Plexin A1 adaptor. Cdk5 and GSK3β also hamper 33 the binding and recruitment of Dynein onto FEME carriers by Bin1. Interestingly, we found that 34 GSK3β binds to Endophilin, thus imposing a local regulation of FEME. Collectively, these findings 35 place the two kinases as key regulators of FEME, licensing cells for rapid uptake by the pathway only 36 upon when Cdk5 and GSK3β activity is low. 38 39Clathrin-mediated endocytosis (CME) is the major uptake pathway in resting cells 1,2 but additional Clathrin-40 independent endocytic (CIE) routes, including fast Endophilin-mediated endocytosis (FEME), perform 41 specific functions or internalize various cargoes 3,4 . FEME is not constitutively active but is triggered upon the 42 stimulation of selected cell surface receptors by their ligands 5 . These include G-protein coupled receptors 43 (e.g. β1-adrenergic receptor, hereafter β1AR), receptor tyrosine kinases (e.g. epidermal growth factor 44 receptor, EGFR) or cytokine receptors (e.g. Interleukin-2 receptor) 5 . In resting cells, FEME is primed by a 45 cascade of molecular events starting with active, GTP-loaded, Cdc42 recruiting CIP4/FBP17 that engage the 46 5'-phosphatase SHIP2 and Lamellipodin (Lpd). The latter then concentrates Endophilin into clusters on 47 discrete locations of the plasma membrane 6 . In absence of receptor activation, the clusters dissemble 48 quickly (after 5 to 15 sec) upon local recruitment of the Cdc42 GTPase-activating proteins RICH1, SH3BP1 49 or Oligophrenin 6 New priming cycles start nearby, constantly priming the plasma membrane for FEME. Upon 50 activation, receptors are quickly sorted into pre-existing Endophilin clusters that then bud to form FEME 51 carriers, which are Clathrin-negative, Endophilin-positive assemblies (EPAs) found in the cytosol. The en...

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“…Among the various clathrin-independent transcytotic pathways (Tuma and Hubbard, 2003; Sandvig et al, 2018), caveolae are particularly abundant in vascular endothelial cells (Frank et al, 2003). Caveolae are caveolin-coated 50-100 nm flask-shaped invaginations of the plasma membrane (Palade, 1953; Palade, 1961).…”

Section: Discussionmentioning

confidence: 99%

Suppression of transcytosis regulates zebrafish blood-brain barrier development

O’Brown

2019

Preprint

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24As an optically transparent model organism with an endothelial blood-brain barrier (BBB), 25 zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental 26 profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms 27 governing the zebrafish BBB remain poorly characterized. Here we find a spatiotemporal gradient 28 of barrier acquisition. Moreover, we capture the dynamics of developmental BBB leakage using 29 live imaging, revealing a combination of steady accumulation in the parenchyma and sporadic 30 bursts of tracer leakage. Electron microscopy studies further reveal that this steady accumulation 31 results from high levels of transcytosis that are eventually suppressed, sealing the BBB. Finally, 32we demonstrate a key mammalian BBB regulator Mfsd2a, which inhibits transcytosis, plays a 33 conserved role in zebrafish. Mfsd2aa mutants display increased larval and adult BBB permeability 34 due to increased transcytosis. Our findings indicate a conserved developmental program of 35 barrier acquisition between zebrafish and mice.36 109 leakage of low molecular weight tracers 1 kDa and below into the brain parenchyma (Nitta et al.,110 2003; Campbell et al. 2008; Sohet et al., 2015; Yanagida et al. 2017). At 3 dpf, we observed a 111 sealed barrier in the hindbrain as previously described (Jeong et al., 2008), with only a few of the 112 parenchymal cells taking up the injected tracer (average of 2 ± 0.3 cells/embryo with NHS and 2 113 ± 0.4 cells/embryo with Dextran; Figure 1 -Supplement 1), which we quantify as a proxy of tracer 114 6 leakage into the brain. However, in the midbrain we observed an increased number of 115 parenchymal cells that accumulated the circulating tracers (average of 24 ± 1 cells/embryo with 116 NHS and 24 ± 1 cells/embryo with Dextran; Figure 1C and 1D), indicating that the tracers leaked 117 out of the blood vessels into the brain and that the midbrain barrier is not yet functional. In addition 118 to the use of exogenous injected fluorescent tracers, we also assayed BBB permeability with an 119 endogenous transgenic serum DBP-EGFP fusion protein (Tg(l-fabp:DBP-EGFP)) to account for 120 injection artifacts (Xie et al., 2010). At 3 dpf, we observed similar leakage patterns with the 121 transgenic serum protein as we did with the injected tracers (average of 24 ± 1 cells/embryo in 122 the midbrain and average of 2 ± 0.4 cells/embryo in the hindbrain; Figure 1C and D; Figure 1 -123 Supplement 1). At 4 dpf, the BBB in the hindbrain is completely functional with few tracer-filled 124 parenchymal cells (average of 3 ± 0.4 cells/embryo with NHS, 2 ± 0.4 cells/embryo with Dextran 125 and DBP-EGFP; Figure 1 -Supplement 1). However, the midbrain BBB remains leaky (average 126 of 23 ± 1 cells/embryo with NHS and DBP-EGFP and 24 ± 1 cells/embryo with Dextran; p=0.68 127 compared to 3 dpf, one-way ANOVA; Figure 1C and 1D). However, at 5 dpf the number of 128 midbrain parenchymal cells that uptake the tracers is dramatically reduced ...

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Clathrin-independent endocytosis: an increasing degree of complexity

Cited by 163 publications

References 110 publications

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

Suppression of transcytosis regulates zebrafish blood-brain barrier development

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