Characterization of erythrocyte membrane domains using fluorescence digital imaging microscopy

Fluorescence digital imaging microscopy was used to characterize the lateral distribution of the lipid and protein components of fluorescently labeled erythrocyte membranes. Experiments with erythrocytes and erythrocyte ghosts labeled with fluorescent phospholipids showed that the lipids are distributed laterally into areas of enrichment, or domains. Double-labeling experiments of ghosts using NBD- and Dansyl-labeled phospholipids demonstrated that the separate phospholipids are distributed differently in the membrane, with corresponding unequal enrichment in the domains. Time-course and temperature experiments of labeled ghosts showed that the domains are stable with time and over a broad range of temperatures. Leaflet-directed labeling also demonstrated that the lipid distributions differ in the separate leaflets. Nonspecific labeling of the erythrocyte membrane proteins showed that the proteins are also distributed into domains. Double-labeling experiments demonstrated that the areas of protein and lipid enrichment frequently coincide, thus suggesting that the domains arise from interactions between the membrane proteins and lipids. Ghosts double-labeled with the fluorescent Band 3 affinity label SITS and fluorescent phospholipids were used to show that the size and enrichment of the lipid and protein domains depended principally on the ionic strength, but not the temperature (below 37$\sp\circ$C), of the sample. Separate double-labeling experiments also revealed that there is a specificity in the lipid enrichment with the Band 3 domains. SITS-labeled ghosts immunofluorescently stained for spectrin revealed that the areas of Band 3 enrichment coincide with areas of spectrin enrichment. Also, spectrin aggregation by antibody crosslinking increased the Band 3 enrichment in the domains. However, trypsin or chymotrypsin treatment of ghosts revealed that the separate Band 3 fragments could still form domains in the absence of the cytoskeleton. However, a fluorescent phospholipid double-labeling experiment of chymotrypsin treated ghosts showed that the lipids were now evenly distributed in the membrane relative to untreated ghosts. Overall, these experiments characterize the lateral heterogeneity of the lipid and protein components in the erythrocyte membrane.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Rodgers, William Allen

Contributors dc:contributor

• Glaser, Michael

• Biology, Cell
• Chemistry, Biochemistry
• Biophysics, General

Statement dc:rights

• Copyright 1992 Rodgers, William Allen

Language dc:language

eng

Identifier

AAI9215880
(UMI)AAI9215880

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21708