Phase separation of Ede1 promotes the initiation of endocytic events

Kozak, Mateusz; Kaksonen, Marko

doi:10.1101/861203

Cited by 18 publications

(19 citation statements)

References 62 publications

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“…These results agree with the apparent requirement for both Fcho1 and Eps15 to drive efficient endocytosis 3,6 . Notably, recent work by Kozak and Kaksonen 44 showed that the Saccharomyces cerevisiae homolog of Eps15, Ede1, can also form phase separated condensates in yeast cells, in agreement with our results.…”

Section: Discussionsupporting

confidence: 93%

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: Discussionsupporting

confidence: 93%

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Day

Kago

Wang

et al. 2019

Preprint

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“…Collectively, these data suggest that END sites are dynamic compartments containing an ensemble of endocytic proteins exhibiting hallmarks of LLPS. Notably, this conclusion is corroborated by a study posted on bioRxiv during the course of our manuscript preparation (Kozak and Kaksonen, 2019).…”

Section: Ends Exhibit Hallmarks Of Llpssupporting

confidence: 74%

“…In case of endocytosis, it has been suggested that LLPS of endocytic proteins serves as a principal mechanism by which the complex assembly of CME proteins during endocytosis is achieved (Bergeron-Sandoval et al, 2018;Day et al, 2019;Kozak and Kaksonen, 2019). What restricts liquid-liquid phase separation of CME proteins during regular endocytosis and inhibits END formation?…”

Section: Discussionmentioning

confidence: 99%

A selective autophagy pathway for phase separated endocytic protein deposits

Wilfling

Lee

Erdmann

et al. 2020

Preprint

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“…A liquid phase of proteins is generated through weak hydrophobic interactions. Previous studies demonstrate that 1,6-hexanediol, an aliphatic alcohol, can disrupt such interactions and dissolve a variety of liquid droplets including stress granules, P bodies, and endocytic proteins (Gopal et al, 2017;Kroschwald et al, 2015;Patel et al, 2007;Kozak and Kaksonen, 2019;Wilfling et al, 2020).…”

Section: Dyn1xa Puncta Are Phase-separatedmentioning

confidence: 99%

Dynamin is primed at endocytic sites for ultrafast endocytosis

Imoto

Raychaudhuri

Fenske

et al. 2021

Preprint

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Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by ~100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

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Phase separation of Ede1 promotes the initiation of endocytic events

Cited by 18 publications

References 62 publications

Liquid-like protein interactions catalyze assembly of endocytic vesicles

Liquid-like protein interactions catalyze assembly of endocytic vesicles

A selective autophagy pathway for phase separated endocytic protein deposits

Dynamin is primed at endocytic sites for ultrafast endocytosis

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