Comparing Novel Techniques for Measuring the Bending Modulus of Lipid Bilayer Membranes in Erythrocytes

Open Access
Author:
Frazzette, Nicholas James
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Peter J Butler, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Herbert Herling Lipowsky, Faculty Reader
Keywords:
  • mechanobiology
  • bending modulus
  • mechanotransduction
  • lipid bilayers
  • membrane bending
  • erythrocytes
  • TCSPC
  • fluorescence
Abstract:
Cellular mechanobiology is the area of biology in which mechanical forces are sensed by cells, and transduced into intracellular biochemical signals, leading to changes in cell function. Cells interact with their environments principally through the cell membrane, where lipids, proteins, and other receptors transmit mechanical and chemical signals to the cell interior. More recently, research has shown that as the lipid bilayer membrane bends or stretches, protein activation can occur as areal changes can induce protein conformational changes or changes in protein clustering. Critically, therefore, understanding the energies and forces that determine membrane bending will help understand the nature of mechanotransduction and its associated cell signaling. The erythrocyte membrane is a popular model membrane of study due to the known flexibility of erythrocytes as they flow through the vasculature. In this thesis, a novel method is presented of determining membrane bending modulus and area-per-lipid using lifetime fluctuations of embedded fluorophores. As the membrane bends and flexes, the area around individual lipids or fluorescent lipid analogs is reduced or expanded due to compression or tension, respectively. Because the fluorescence lifetime of many fluorophores is strongly regulated by the immediate local environment of the fluorophore, these areal changes lead to detectable lifetime changes. Using this method, it is shown that the bending modulus of healthy erythrocytes is approximately 55.60 kBT, in good agreement with commonly obtained literature values, and the area per lipid is approximately 44.22 Å2, a previously unreported value because it is inaccessible with currently available techniques. These values were also experimentally validated against a novel, low power optical trapping method for detecting membrane mechanics. Importantly, the use of fluorescence lifetime fluctuations represents a simple and versatile technique that can be applied to many cell solution states, such as varying pH or osmolarity, as well as many different types of cells.