Visualizing Membrane Transformation in Live Cell with STED

Membrane fusion and budding mediate fundamental biological processes like intracellular trafficking, exocytosis, and endocytosis. For many decades, fusion has been thought to open a nanometer-range pore that may subsequently close or dilate irreversibly, whereas budding transforms flat membranes into vesicles. This classical view has been difficult to verify owing to the difficulty of seeing membrane transformation in real time.

In this webinar, Dr. Wu will present a newly developed workflow to visualize in real-time the membrane transformations of exo- and endocytosis in live cells (neuroendocrine chromaffin cells). Real-time visualization reveals membrane transformations of exo- and endocytosis far exceeding the classical view. He synthesizes a new model of exo- and endocytosis based on real-time observations and describes its underlying mechanistic principles and functions.

In this new model, 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 W-shaped profiles pre-formed through the flat-to-L-to-W-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 molecular mechanics, and functions may be conserved for fusion and budding in general.

Learning Objectives

• Examine exocytotic vesicle membrane and fusion pore dynamics and their underlying mechanics.
• Demonstrate endocytic transformation from flat to L-, W-, and O-shape and underlying mechanics.
• Discuss a workflow for visualizing vesicle fusion and budding in real-time.

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