De novo endocytic clathrin coats develop curvature at early stages of their formation

Willy, Nathan M.; Ferguson, Joshua P.; Akatay, Ata; Huber, Scott D.; Djakbarova, Umidahan; Silahli, Salih; Cakez, Cemal; Hasan, Farah; Chang, Henry; Travesset, Alex; Li, Siyu; Zandi, Roya; Liu, Dong; Betzig, Eric; Cocucci, Emanuele; Kural, Cömert

doi:10.1016/j.devcel.2021.10.019

Cited by 36 publications

(62 citation statements)

References 66 publications

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“…6 ). In agreement with previous research, we found the beginning of coat curvature can occur following the initiation of clathrin accumulation 12 , 55 . Additionally, we identified a population of endocytic events developing curvature prior to detectable accumulation of clathrin.…”

Section: Discussionsupporting

confidence: 93%

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

et al. 2022

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Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.

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Section: Discussionsupporting

confidence: 93%

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

et al. 2022

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“…1, B and C), the FCHO2 ring expands further. The clathrin signal also appears annular, presumably due to the Z-projection of the spheroidal dome it must form ( 34 , 37 , 38 ) and the exponentially decaying intensity of the incident light within evanescent TIR field.…”

Section: Resultsmentioning

confidence: 99%

“…It would then resist further expansion, and the resulting maximum diameter of the annulus should help define the maximum size of a CCP/CCV by the number of clathrin adaptors it can corral inside it, providing an explanation for why all CCPs in the RPE and U-2 OS cells are basically of the same size/intensity. It is therefore possible that the amount of FCHO/Eps15 may play a role in defining the standard CCV size/curvature along with the amount of CALM ( 38 , 65 ). An annular structure has also been seen in the endocytic CCPs of yeast ( 66 ), despite its endocytosis being largely actin dependent and so mechanistically different, suggesting that not only this design feature is conserved throughout eukaryotes but also its formation may be a common mechanism in many transport vesicle genesis events.…”

Section: Discussionmentioning

confidence: 99%

FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P ₂ -dependent switch

et al. 2022

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Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence–structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2’s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP’s outer edge to engage and activate further AP2s to drive CCP growth/maturation.

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“…1B,C) the FCHO2 ring expands further. The clathrin signal also appears annular, presumably due to the z projection of the spheroidal dome it must form ( 36, 37 ) and the exponentially decaying intensity of the incident light within evanescent TIR field.…”

Section: Resultsmentioning

confidence: 99%

“…It would then resist further expansion and the resulting maximum diameter of the annulus should help to define the maximum size a CCP/CCV by the number of clathrin adaptors it can corral inside it, providing an explanation for why all CCPs in the RPE and U-2 OS cells are basically of the same size/intensity. It is therefore possible that the amount of FCHO/Eps15 may play a role in defining the standard CCV size/curvature along with the amount of CALM (37,65) Intriguingly an annular structure has also been seen in the endocytic CCPs of yeast (66), despite its endocytosis being largely actin dependent and so mechanistically different, suggesting that this design feature is not only conserved throughout eukaryotes but also that its formation may be a common mechanism in many transport vesicle genesis events To conclude, our eTIRF SIM shows that a small number of FCHO molecules, presumably clustered on the PM by virtue of their FBAR-domains binding PtdIns4,5P2 and PS and by forming phase-separated nano clusters with Eps15 ((17) (38) and Figs. 1D,E) act as potential sites for CCP nucleation.…”

Section: Possible Roles For the Fcho/eps15 Corralmentioning

confidence: 99%

FCHO controls AP2’s critical endocytic roles through a PtdIns4,5P₂ membrane-dependent switch

Zaccai

Kadlecova

Dickson

et al. 2022

Preprint

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Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrincoated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

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De novo endocytic clathrin coats develop curvature at early stages of their formation

Cited by 36 publications

References 66 publications

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P ₂ -dependent switch

FCHO controls AP2’s critical endocytic roles through a PtdIns4,5P₂ membrane-dependent switch

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De novo endocytic clathrin coats develop curvature at early stages of their formation

Cited by 36 publications

References 66 publications

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P 2 -dependent switch

FCHO controls AP2’s critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

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FCHO controls AP2’s initiating role in endocytosis through a PtdIns(4,5)P ₂ -dependent switch

FCHO controls AP2’s critical endocytic roles through a PtdIns4,5P₂ membrane-dependent switch