Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis

Abstract

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

Cell Membranes Resist Flow

“…Membrane tension affects cell migration (Gauthier et al, 2011; Houk et al, 2012; Keren et al, 2008; Mueller et al, 2017), vesicle fusion and recycling (Boulant et al, 2011; Gauthier et al, 2011; Maritzen and Haucke, 2017; Masters et al, 2013; Shillcock and Lipowsky, 2005; Shin et al, 2018; Thottacherry et al, 2017; Wen et al, 2016), the cell cycle (Stewart et al, 2011), cell signaling (Basu et al, 2016; Groves and Kuriyan, 2010; Houk et al, 2012; Huse, 2017; Romer et al, 2007), and mechanosensation (He et al, 2018; Phillips et al, 2009; Ranade et al, 2015). However, there has been controversy over the speed and degree to which localized changes in membrane tension propagate in cells (Diz-Muñoz et al, 2013).…”

Correlative SICM‐FCM reveals changes in morphology and kinetics of endocytic pits induced by disease‐associated mutations in dynamin

“…Collectively, the above limitations resulted in remarkable heterogeneity of putative CCPs because fluorescence signals from clathrin contained in genuine CCPs, flat lattices, and endosomes are difficult to distinguish (29, 30). Recently, developments in fluorescence probes for phospholipid components of cell membranes have enabled detailed characterization of vesicle fusion and fission kinetics and helped to elucidate the role of dynamin in fusion pore dynamics in live cells at ∼60 nm spatial and subsecond temporal resolution using STED (31, 32). Also, LLSM was used to follow membrane remodelling and track CCPs and dynamin in live cells at 5.7 s per frame temporal and 150 × 280 nm xz resolution (33, 34).…”

Direct Visualization of Wide Fusion-Fission Pores and Their Highly Varied Dynamics

“…Live-Cell Imaging of the Chromaffin Cell Fusion-Fission Pore (A) Schematic of the experimental paradigm used in Shin et al (2018). Adrenal chromaffin cells were Imaged with STED microscopy.…”

“…EM images give the best spatial resolution but only provide one snapshot of a secretory event. Now, in this issue of Cell, Shin et al present the first live-cell dynamic imaging of a fusion pore (Shin et al, 2018). Using super-resolution microscopy, they show that a fusion pore can be significantly larger and longer-lived than previously thought and that its varied dynamics confer at least four types of post-fusion responses.…”

“…Shin et al went on to investigate the opposite process, membrane expansion, which has been associated with actin-dependent membrane tension. Addition of Latruncu- lin A, which hampers filamentous actin polymerization and thus generation of membrane tension, limited expansion of the fusion pore (Shin et al, 2018). …”