Bilayer Formation on Protocells

Open Access
Author:
Grigas, Alex T
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Christine Dolan Keating, Thesis Supervisor
  • Lorraine C Santy, Honors Advisor
  • Philip C Bevilacqua, Thesis Supervisor
  • Wendy Hanna-Rose, Faculty Reader
Keywords:
  • coacervates
  • phospholipid bilayer formation
  • protocell models
  • RNA World
  • liposome assembly
Abstract:
One of the prevailing theories of the origin of life postulates an RNA world that existed before life arose, in which RNA stored information and carried out catalysis. However, in order for a dilute ocean of simple RNAs to yield anything like a cell, these important biological molecules and ions would first have to be highly concentrated and protected. One such mechanism involves compartmentalization via liquid-liquid phase separation (LLPS). In this study, we focused on a particular type of LLPS called complex coacervation. When oppositely charged polyelectrolytes are in solution together, they will often phase separate into liquid droplets, which are highly concentrated in polyelectrolyte, and a dilute bulk water phase. Complex coacervation is of particular interest for the RNA world because, as RNA is a highly negatively charged polymer, it can be concentrated through coacervation. Another necessary condition for cellular life besides concentration is a highly selective membrane. Phospholipid bilayers not only protect cells from their environment, they also make selective transport possible, which is necessary in order to create gradients and drive energy production. That is why in this study we focused on the interactions between phospholipids formed into liposomes and different coacervate systems formed using different charge density molecules at different charge ratios. The coacervates were characterized through microscopy, their absorption and their surface charge. The liposomes were characterized through zeta potential as well as dynamic light scattering. Then, the interactions between the liposomes and coacervates were observed using fluorescent microscopy. Fluorescent recovery after photobleaching (FRAP) was then used in order to investigate the extent of bilayer formation. We observed three different types of interactions: liposome assembly at the interface, aggregation of liposomes at the interface, and the aggregation of liposomes. FRAP data showed no recovery after 10 minutes, which indicates a bilayer was not formed and that the liposomes’ lateral movement is restricted by the interface. This has to do with the strength of the interaction between the liposome and the coacervate surface as well as the liposome and the free polyelectrolyte in the solvent. While no bilayer formation was observed, the many systems that resulted in liposome assembly at the interface are a positive indication of the possibility of bilayer formation under different conditions and to the continued viability of coacervates as a protocell model.