Membrane transformations of fusion and budding

Abstract

Membrane fusion and budding mediate fundamental processes like intracellular trafficking, exocytosis, and endocytosis. Fusion is thought to open a nanometer-range pore that may subsequently close or dilate irreversibly, whereas budding transforms flat membranes into vesicles. Reviewing recent breakthroughs in real-time visualization of membrane transformations well exceeding this classical view, we synthesize a new model and describe its underlying mechanistic principles and functions. Fusion involves hemi-to-full fusion, pore expansion, constriction and/or closure while fusing vesicles may shrink, enlarge, or receive another vesicle fusion; endocytosis follows exocytosis primarily by closing Ω-shaped profiles pre-formed through the flat-to-Λ-to-Ω-shape transition or formed via fusion. Calcium/SNARE-dependent fusion machinery, cytoskeleton-dependent membrane tension, osmotic pressure, calcium/dynamin-dependent fission machinery, and actin/dynamin-dependent force machinery work together to generate fusion and budding modes differing in pore status, vesicle size, speed and quantity, controls release probability, synchronization and content release rates/amounts, and underlies exo-endocytosis coupling to maintain membrane homeostasis. These transformations, underlying mechanisms, and functions may be conserved for fusion and budding in general.

Fig. 1. Classical exo-endocytosis model.

a Full-collapse fusion. I, EM images showing the hypothesized sequence of full-collapse fusion (from upper left to upper right, then lower left to lower right). Images were taken from neuromuscular junctions that were frozen 3.7, 5.2, 5.2, 5.2, 20 ms, and 50 ms after stimulation. These data led to a full-collapse fusion proposal. Reproduced from J E Heuser, T S Reese; Structural changes after transmitter release at the frog neuromuscular junction. J Cell Biol 1 March 1981; 88 (3): 564–580. 10.1083/jcb.88.3.564. II Cell-attached recordings of amperometric current (Amp), vesicular membrane capacitance (C_v), and fusion pore conductance (G_p) at a chromaffin cell. Reproduced from Albillos, A., Dernick, G., Horstmann, H. et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997). 10.1038/39081. III Cell-attached capacitance recordings of C_v and G_p at the release face of a calyx of Held nerve terminal. Adapted from He, L., Wu, XS., Mohan, R. et al. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444, 102–105 (2006). 10.1038/nature05250. b Kiss-and-run I. EM image of a Ω-shape profile in a frog neuromuscular junction led to kiss-and-run proposal. Reproduced from B. Ceccarelli, W. P. Hurlbut, A. Mauro; DEPLETION OF VESICLES FROM FROG NEUROMUSCULAR JUNCTIONS BY PROLONGED TETANIC STIMULATION. J Cell Biol 1 July 1972; 54 (1): 30–38. 10.1083/jcb.54.1.30. II Cell-attached recordings of Amp, C_v, and G_p at a chromaffin cell. Reproduced from Albillos, A., Dernick, G., Horstmann, H. et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997). 10.1038/39081. III Cell-attached recordings of C_v and G_p during a capacitance flicker at the release face of a calyx of Held nerve terminal. Adapted from He, L., Wu, XS., Mohan, R. et al. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444, 102–105 (2006). 10.1038/nature05250. c Endocytic membrane transformations. I. Freeze-fracture EM images of frog neuromuscular junctions arranged in this order to illustrate the hypothetical endocytic process starting from a shallow pit to a vesicle-like Ω-profile. Reproduced from T M Miller, J E Heuser; Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction. J Cell Biol 1 February 1984; 98 (2): 685–698. 10.1083/jcb.98.2.685. II Cell-attached recordings of C_v and G_p reflecting fission pore closure at a calyx of Held release face. Adapted from He, L., Xue, L., Xu, J. et al. Compound vesicle fusion increases quantal size and potentiates synaptic transmission. Nature 459, 93–97 (2009). 10.1038/nature07860. d Classical exo-endocytosis model. Fusion undergoes kiss-&-run or full-collapse fusion, the latter of which is followed by endocytic flat-to-round transformation at an endocytic zone. Blue dots: vesicular contents before fusion (applies to all figures).

Fig. 2. A dynamic pore model of fusion.

a Schematic diagram for setup and labeling method. I In a chromaffin cell, vesicles are preloaded with FFN511 (blue), PM inner leaflet labeled with overexpressed PH_G (green), extracellular solution labeled with A532 (red), a pipette at the whole-cell voltage-clamp configuration for delivering depolarization and recording calcium currents (I_Ca) and membrane capacitance (C_m). II Fusion dynamics (hemi-fusion, pore opening and expansion) are visualized with three probes: FFN511 for release, PH_G for PM inner leaflet fusion, and A532 for A532-permeable pore. b STED XZ plane images of PH_G (green) and A532 (red) showing hemi-fusion (I) and hemi-fusion followed by hemi-to-full fusion (II). Images taken at times relative to 1-s depolarization as labeled (also applies to cI and dI). Adapted from Zhao, WD., Hamid, E., Shin, W. et al. Hemi-fused structure mediates and controls fusion and fission in live cells. Nature 534, 548–552 (2016). 10.1038/nature18598. c Fusion pore expansion. I STED XZ plane images of PH_G and A532 showing fusion pore opening (Middle, A532-permeable) and expansion (right, visible). Data taken from Cell, 173, W. Shin, L. Ge, G. Arpino, et al. Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis, 934-945, Copyright Elsevier (2018). II Cell-attached recordings of the amperometric current (Amp) and fusion pore conductance (G_P). Reproduced from Albillos, A., Dernick, G., Horstmann, H. et al. The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997). 10.1038/39081. d Fusion pore closure (kiss-and-run). I STED XZ plane images of PH_G and A532 showing fusion pore opening and closure. Pore closure prevents bleached A532 from exchange with fluorescent A532 in the bath, leading to A532 fluorescence dimming. Data were published in Cell, 173, W. Shin, L. Ge, G. Arpino, et al. Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis, 934-945, Copyright Elsevier (2018). II Capacitance (Im) flickers with pore conductance (Re) beyond detection limit from cell-attached recordings at the calyx of Held release face Adapted from He, L., Wu, XS., Mohan, R. et al. Two modes of fusion pore opening revealed by cell-attached recordings at a synapse. Nature 444, 102–105 (2006). 10.1038/nature05250. III Cell-attached recordings of a capacitance flicker with a fast amperometric spike at a chromaffin cell. Reproduced from Alés, E., Tabares, L., Poyato, J. et al. High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat Cell Biol 1, 40–44 (1999). 10.1038/9012. e Schematics of fusion pore dynamics and underlying mechanisms. Fusion undergoes hemi-fusion, hemi-to-full fusion, pore expansion, constriction and closure. The dynamics of each of these transitions are the net outcome of competition between fusion machinery (M_Fus), plasma membrane tension and dynamin. Calcium influx triggers vesicle fusion, fusion pore constriction and closure. Hemi-fusion generates no release; small and large fusion pore generates slow and fast release, respectively. Delayed hemi-to-full fusion causes asynchronized release.

Fig. 3. Seven fusion modes differed in pore status and vesicle size.

a Shrink fusion. I–II STED XZ plane images of PH_G (green) and A532 (red) showing shrink fusion: fusion-generated Ω-profiles may shrink until undetectable (I), or until becoming a Λ-shape at the very end of the shrinking process (II). Images taken at times labeled. Data were published in Cell Report, 30, W. Shin, G. Arpino, S. Thiyagarajan, et al. Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents, 421–431, Copyright Elsevier (2020). III. Confocal XZ plane images showing a huge GFP-filled salivary gland vesicle undergoing shrinking at times labeled. Scale bar: 5 µm. Reproduced from Rousso, T., Schejter, E. & Shilo, BZ. Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network. Nat Cell Biol 18, 181–190 (2016). 10.1038/ncb3288. b Enlarge fusion. I STED XZ plane images of PH_G (green) and A532 (red) showing enlarge fusion: the fusion-generated Ω-profile enlarged over time as labeled. Data were published in Cell Report, 30, W. Shin, G. Arpino, S. Thiyagarajan, et al. Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents, 421-431, Copyright Elsevier (2020). II Capacitance flickers with the down-step equal to (left) or larger than (middle and right) the up-step. Reproduced from J.R. Monck, T.G. Alvarez de, J.M. Fernandez, Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore. Proc. Natl. Acad. Sci. USA 87, 7804–7808 (1990). Middle and right panel are consistent with enlarge-close fusion. c Schematic diagram describing seven fusion modes: stay, close, enlarge-stay, enlarge-close, shrink-stay, shrink-close, and shrink fusion. Shrink-related fusion events are associated with a larger pore to generate fast release; enlarge-related fusion events are associated with a smaller pore to generate slow release. Release traces are taken from the article published in Cell Report, 30, W. Shin, G. Arpino, S. Thiyagarajan, et al. Vesicle shrinking and enlargement play opposing roles in the release of exocytotic contents, 421-431, Copyright Elsevier (2020). d I Predicted shrink fusion sequence. Computed vesicle shapes and free energies for squeezing pressure ΔP = 100 Pa and the indicated effective vesicle diameter (D). A transition from Ω- to Λ-shape occurs at D = 56 nm. Data were published in Cell Report, 30, W. Shin, G. Arpino, S. Thiyagarajan, et al. Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents, 421-431, Copyright Elsevier (2020). II Schematic diagram showing the osmotic pressure difference between the intracellular and the extracellular solution (ΔP) squeezes and thus deflates the vesicle, abolishing the vesicular membrane tension and allowing for the vesicular membrane to be reeled into the plasma membrane by the high plasma membrane tension and the actin cortex. Drawing taken from the article published in Cell Report, 30, W. Shin, G. Arpino, S. Thiyagarajan, et al. Vesicle Shrinking and Enlargement Play Opposing Roles in the Release of Exocytotic Contents, 421–431, Copyright Elsevier (2020).

Fig. 4. Sequential compound fusion.

a Three sulphorhodamine-B-filled spots (red) occurred sequentially at the indicated time that may reflect sequential compound fusion of zymogen granules in a pancreatic acinus cell bathed with sulphorhodamine-B. Reproduced from Nemoto, T., Kimura, R., Ito, K. et al. Sequential-replenishment mechanism of exocytosis in pancreatic acini. Nat Cell Biol 3, 253–258 (2001). 10.1038/35060042. b Sequential compound fusion and release visualized in chromaffin cells. I STED XZ plane images of PH_G (green) and A532 (red) showing sequential compound fusion at times indicated from a chromaffin cell (see also cartoon explanations). Adapted from L. Ge, W. Shin, G. Arpino, L. Wei, C. Y. Chan, C. K. E. Bleck, W. Zhao, L. G. Wu, Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells. Sci. Adv. 8, eabm6049 (2022). © The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/. II. STED XZ plane images of PH_G (green) and FFN511 (magenta) showing sequential compound release (two circles) at times indicated from a chromaffin cell (see also cartoon explanations). Adapted from L. Ge, W. Shin, G. Arpino, L. Wei, C. Y. Chan, C. K. E. Bleck, W. Zhao, L. G. Wu, Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells. Sci. Adv. 8, eabm6049 (2022). © The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/. c Schematic diagram depicting membrane transformations of sequential compound fusion, sequential compound kiss-and-run, and compound fusion. New release site formation at the 1st fused vesicle enables sequential compound fusion; 2nd fusion generates asynchronized release; sequential compound fusion or compound fusion may generate large Ω-profiles and vesicles.

Fig. 5. Endocytic membrane transformation, underlying mechanical forces, and function in generating diverse endocytic modes.

a Schematic diagram showing how to visualize endocytic membrane transformation using two probes: PH_G (green) for labeling the PM inner leaflet and A532 (red) for labeling bath solution (bath dye). b STED XZ plane images of PH_G (green) and A532 (red) showing Flat→Λ (I), Λ→Ω (II), Ω→O (III) and Flat→Λ→Ω→O transition (IV). Images taken at times labeled. Data in panel I were adapted from Shin, W., Zucker, B., Kundu, N. et al. Molecular mechanics underlying flat-to-round membrane budding in live secretory cells. Nat Commun 13, 3697 (2022). 10.1038/s41467-022-31286-4. Data in panels II-IV were from the article published in Neuron, 109, W. Shin, L. Wei, G. Arpino, et al. Preformed Omega-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells, 3119–3134, Copyright Elsevier (2021). c I STED XZ plane images of PH_G (green) and Lifeact-mTFP1 (red, labeling F-actin) showing spike-like PH_G-labeled membrane protrusion attached to growing F-actin filaments while the Λ-profile is growing. Adapted from Shin, W., Zucker, B., Kundu, N. et al. Molecular mechanics underlying flat-to-round membrane budding in live secretory cells. Nat Commun 13, 3697 (2022). 10.1038/s41467-022-31286-4. II STED XZ plane images of PH_G (green) and dynamin-mTFP1 (red) showing dynamin puncta flanked and moved with constricting Λ’s base and constricting Ω’s pore—dynamin constricts Λ’s base and Ω’s pore. Adapted from Shin, W., Zucker, B., Kundu, N. et al. Molecular mechanics underlying flat-to-round membrane budding in live secretory cells. Nat Commun 13, 3697 (2022). 10.1038/s41467-022-31286-4. d Schematic diagram showing the Flat→Λ→Ω→O transition mediated by two forces, the F-actin- and dynamin-dependent pulling force and dynamin-dependent constriction force. Calcium is the trigger for each transition, including Flat→Λ, Λ→Ω and Ω→O. The probability of each transition is low as labeled (0.12–0.24). e Pore closure of preformed Ω-profiles and fusion pores, but not flat-to-round transformation, is the main driving force underlying diverse modes of endocytosis, such as ultrafast, fast, slow, compensatory, and overshoot endocytosis that follow depolarization-induced exocytosis. Depol: depolarization; Cm: membrane capacitance, Exo: exocytosis (Cm increase); endo: endocytosis (Cm decay); blue dots: vesicular contents before fusion.

Fig. 6. A new exo-endocytosis membrane transformation model synthesized from live-cell observations.

A schematic diagram showing a new exo-endocytosis membrane transformation model and molecular mechanical mechanisms underlying each membrane transition (M_Fus: fusion machinery). Hemi-fusion generates no release (blue dots: vesicular contents); enlarge-related fusion generates slow release; shrink-related fusion generates fast release. Delayed hemi-to-full fusion or sequential compound fusion generates asynchronized release. Compound fusion generates a large quantal size. Narrow fusion pore may cause partial vesicular content release. Redefined kiss-and-run (including close, enlarge-close and shrink-close fusion, left dash square) and preformed Ω-profile pore closure (right dash square) are major mechanisms underlying diverse endocytic modes, including ultrafast, fast, slow, compensatory, overshoot, and bulk endocytosis.