Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids.

Abstract

Abstract. Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confnement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-rim colloidal gold conjugated to anti-fluorescein (anti-H). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (Da) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 • 10 -9 cm2/s. These values were lower than the average diffusion coefficients (Dr) (5.4 to 9.5 x 10 -9 cm2/s) obtained by FRAP. The lower D6 is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased Do to 2.8 x 10 -9 cm2/s, but did not affect Dr.Pericellular matrix viscosity was estimated from the frictional coefficients computed from DQ and Dr and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise.To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacemerit (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 #m in diameter.

Evaluation of membrane physiology following fluorescence activated or magnetic cell separation

“…Consequently, future investigations on membrane fluidity should include constraints to lateral diffusion such as peripheral membrane proteins as proposed by Eisinger and Scarlata (45) or Jacobsen et al (46). New technical approaches to be taken into account are fluorescence recovery after photobleaching using the confocal microscope (47,48) or video-enhanced microscopy utilizing colloidal gold-targeted lipid molecules (14).…”

Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells.

“…Cell membrane was labeled with N-(5-fluorescein thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE, Molecular Probes) following the procedure of Lee et al (1993).…”

Diffusion-limited reactions in G-protein activation: Unexpected consequences of antagonist and agonist competition

“…For the two dimensional reactions in the cell membrane, G-protein activation and receptor phosphorylation, we used values of D = 10 −11 -10 −9 cm 2 /sec (Lee et al, 1993;Sako and Kusumi), SA = 1000 μm 2 , and [G] = 10 -100/μm 2 (Pardo et al, 1997). For G-protein activation we then calculate range for k + of 10 −6 to 10 −3 (#/cell) −1 sec −1 .…”