Molecular dynamics of biomembrane as probed by multifaceted biophotonics

Open Access
Author:
Kyoung, Minjoung
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
November 30, 2007
Committee Members:
  • Erin Elizabeth Sheets, Committee Chair
  • Christine Dolan Keating, Committee Member
  • Tae Hee Lee, Committee Member
  • Ahmed A Heikal, Committee Member
Keywords:
  • membrane
  • spectroscopy
  • microscopy
  • TIR
  • HOT
  • FCS
  • cell
  • lipid
Abstract:
The cellular membrane plays a vital role in numerous biological processes, such as communication, hormonal response and transport of both small and large molecules into and out of the cell. In these processes, intra-and intermembrane interactions and their dynamics are critical. Any intramembrane reactions depends upon later interactions with the two-dimensional bilayer, which are not yet well understood. However, the underlying mechanism of lateral molecular diffusions in biomembranes is not fully understood. For example, it is unclear whether these molecules diffuse in a controlled manner and if they do, what kinds of interactions and properties govern their molecular dynamics. In contrast, intermembrane interactions are key processes for endocytosis and exocytosis. Small vesicles diffuse close to and fuse with the target membrane or are pinched off and diffuse away from the target membrane. Although much focus has been directed toward the actual fusion and fission steps, understanding of vesicle dynamics and their interactions near the target membrane remain elusive, despite their importance. The major intricacy in studying weak, yet highly cooperative, these dynamics is the complex heterogeneity of cellular membranes, which requires integrated and noninvasive techniques to investigate the requisite broad range of spatial and temporal scales. To date, however, conventional techniques have been optimized to probe membrane dynamics over a narrow range of space and time. To fully understand complex membrane dynamics in vivo, model systems allow systematic and rigorous exploration of physical mechanisms. Therefore, I employed supported planar bilayers and small unilamellar vesicles to study membrane dynamics, under various controlled conditions. To interrogate intra- and intermembrane dynamics, we developed multimodal microscopy that can simultaneously perform several microscopic and spectroscopic techniques, such as prism based- and objective based total internal reflection (TIR) microscopy, fluorescence correlation spectroscopy (FCS), single particle tracking (SPT) and holographic optical trapping. Individual techniques were validated by proof-of-concept experiments including imaging local adhesion of C3H 10T½ fibroblast, monitoring the diffusion properties of lipid analogs in the plasma membrane of RBL cells, tracking individual lipids in model membrane systems and trapping arrays of silica beads (~ 0.8 um in diameter) in defined patterns. By combining three different methods, i.e., TIR, FCS and optical trapping, I simultaneously manipulated and probed the dynamics of individual particles near a substrate-solution interface. I observed the diffusion of polystyrene particles and trapped single particles (~84 nm in diameter) near glass surfaces treated with several materials, such as polyethylene glycol 8000, bovine serum albumin or sodium hydroxide. These results indicated that particle diffusion is influenced by surface interactions, which may have further implications in vesicle/membrane interactions that subsequently were investigated. To provide a comprehensive interpretation of endocytosis and exocytosis, the dynamics of freely diffusing small vesicles near supported planar bilayers were investigated using TIR-FCS. The population distributions of vesicles near planar bilayers were affected by ionic strength, pH of surrounding buffers, as well as lipid compositions of the planar bilayers. As a result, vesicles experienced altered hydrodynamic interactions with the planar bilayers and thus changing measured vesicle diffusion near the bilayers. These experimentally determined vesicle dynamics agree with theoretical simulations. Based on these results, I propose that, when small vesicles are not in contact with cellular membrane, they do not randomly diffuse around membranes, but may diffuse in a controlled manner under various dynamic biological conditions. To further mimic the cellular environment, the interactions of vesicles with supported planar bilayer were investigated under crowded and highly viscous conditions. By adding glycerol to the small vesicle/planar membrane model system, the dynamics of vesicles near planar bilayer were considerably reduced. Increasing the solution viscosity with glycerol enhanced the attractive interactions of vesicles with planar bilayers. In addition, I observed that Ficoll 70 solutions that increased viscosity and the excluded volume effect can further reinforce the attractive interactions between vesicles and planar bilayers. The excluded volume effect of Ficoll 70 slightly enhanced vesicle/membrane interactions, in contrast to the large viscosity effects induced by Ficoll 70. In addition, vesicles showed more dramatic changes in their interactions with negatively charged planar bilayer than with neutral planar bilayer. Therefore these results imply that, within cells, vesicle interactions are improved mainly due to viscosity effects.