Theses Doctoral

Membrane Rupture, Membrane Fusion and the Regulation of Exocytosis

An, Dong

Biological membranes form the structural boundaries and compartments of cells, owing to their robustness and impermeability facilitated by phospholipid bilayers. The strength of biological membranes is intricately linked to the behavior of membrane pores, whose formation and expansion can lead to membrane rupture. However, processes essential for drug delivery, gene editing via genetic material transfer, and antimicrobial peptide action necessitate controlled membrane disruption for efficient cellular entry. Likewise, fundamental phenomena such as exocytosis, including neurotransmitter release between neurons and hormone secretion for physiological responses, rely on membrane breach to release cargo beyond cell confines. Exocytosis involves the fusion of cargo-contained vesicle membranes with the cell's plasma membrane, resulting in the release of cargo into the extracellular milieu. Post-release, these fused vesicles may either integrate with the plasma membrane, remain stationary, enlarge, or depart the release site through fusion pore closure, which, in turn, can modulate exocytosis rate through site availability. However, the precise mechanism of membrane rupture remains elusive. Similarly, the pathway of membrane fusion facilitated by SNARE proteins, pivotal in cellular fusion machinery, remains a subject of debate. Additionally, the mechanisms governing exocytosis remain incompletely understood.

To address these inquiries, we employ ultra-coarse-grained molecular dynamics simulations which can explore these phenomena in physiological timescale. These simulations explore membrane rupture mechanisms via pore formation and expansion under varying membrane tension. Furthermore, the research addresses how SNARE proteins drive membrane fusion. In addition, we also rigorously analyze confocal microscopy data from Ling-Gang Wu's research group and develop a quantitative model to elucidate exocytosis rate regulation. Furthermore, the research verifies the robustness of a mathematical model outlining Ca2+-mediated membrane fusion and establishes that hemifusion diaphragms (HDs), where only the outer leaflets of membranes fuse, act as hubs in the Ca2+-mediated fusion network. This finding casts new light on the role of membranes in SNARE-mediated fusion. In the extra study, we analyzed fission yeast contractile ring behavior based on z-stack confocal microscopy data from Mohan Balasubramanian's research group, offering insights into the mechanism behind a critical step in cytokinesis.

Chapter one examines membrane pore energetics and bilayer rupture times through highly coarse-grained simulations operating at submillisecond time scales. No metastable states are detected during pore formation. At lower tensions, small hydrophobic pores mature into large hydrophilic pores that ultimately rupture from reversible hydrophilic pores, aligning with classical tension-dependent rupture times. At higher tensions, membranes rupture directly from small hydrophobic pores, with rupture times exhibiting exponential tension dependence. Upon reaching a minimum hydrophobic pore size, a critical tension threshold prompts immediate rupture. This analysis corroborates established experimental findings but reveals that the high-tension exponential regime is not related to long-lived pre-pore defects but rather to the instability of hydrophilic pores beyond a critical tension, leading to significant changes in pore dynamics and rupture kinetics.

Chapter two describes utilizing ultra-coarse-grained simulations to dissect the core requirements of membrane fusion and unravel the intricacies of SNARE-mediated fusion. Remarkably, simulations conducted on a millisecond timescale expose the inefficiency of fusion through simple body forces pushing vesicles together. Successful inter-vesicle fusion hinges on the rod-like structure of fusogens, ensuring their sufficient length for effective fusion and subsequent clearance from the fusion site via entropic forces. Simulations featuring rod-shaped fusogens and SNARE proteins demonstrate the fusion of 50-nanometer vesicles in submilliseconds, propelled by entropic forces that direct a predictable fusion pathway. The entropic force hypothesis of SNARE-mediated membrane fusion garners strong support from these findings, emphasizing the necessity of the rod-like configuration of the SNARE complexes for entropic force generation and fusion.

Chapter three focuses on the spatiotemporal dynamics of dense-core vesicle exocytosis events in chromaffin cells, deducing a novel mechanism for exocytosis regulation based on the availability of release sites. Repeated fusion supports membrane reservoir comprising incompletely merged or closed vesicles, occupying release sites and dampening exocytosis frequency. Mathematical modeling suggests reservoir formation relies on locally reduced membrane tension, eliminating the driving force for vesicle merging. Endocytosis facilitates the clearance of unmerged vesicles from the reservoir, ultimately restoring release site availability for subsequent exocytosis events.

Chapter four introduces a mathematical model pinpointing the hemifusion diaphragm (HD) as the decision nexus dictating the outcomes of pathways and the fate of final products during multivalent cation-mediated membrane interactions. Transient formation of a high-tension hemifusion interface between membrane-enclosed compartments underscores the model's prediction of fusion, dead-end hemifusion, or vesicle lysis. This comprehensive framework offers predictive insights into interactions mediated by cationic fusogens within membrane-enclosed compartments.

Chapter five offers a unique exploration of writhing contractile rings in fission yeast cell ghosts, resulting from controlled digestion of the cell wall and subsequent membrane permeabilization. This innovative approach unveils the intricate dynamics of contractile rings under exceptional circumstances. Writhing of rings is attributed to the detachment of sections from the weakened membrane, followed by their coiling due to apparent twisting torques at anchoring points. Iterative rotations give rise to multiple coils within the rings.


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More About This Work

Academic Units
Chemical Engineering
Thesis Advisors
O'Shaughnessy, Ben
Ph.D., Columbia University
Published Here
November 8, 2023