Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non‐raft phospholipid molecules (0.33 µm2/second), despite Bdp‐Chol's probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them. A fluorescent cholesterol analogue, Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol), exhibited diffusion at the fastest rate ever found for any membrane molecules in the plasma membrane, with a median diffusion coefficient of 3.4 µm2/second, ∼10× greater than that of phospholipids, despite Bdp‐Chol's probable association with raft domains. Bdp‐Chol and phospholipids diffused at comparable rates in the absence of actin membrane‐skeleton, indicating that Bdp‐Chol hops across the actin‐fence‐pickets' compartment boundaries ∼10× faster than phospholipids. Raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.