Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity.
During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.
Lee
James C-M
JC
Department of Biological Engineering, University of Missouri-Columbia, USA.
Gimm
J Aura
JA
Lo
Annie J
AJ
Koury
Mark J
MJ
Krauss
Sharon W
SW
Mohandas
Narla
N
Chasis
Joel A
JA
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D001842:Bone and Bones / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D004900:Erythroblasts / Q000166:cytology*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D006021:Glycophorins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D050356:Lipid Metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D012156:Reticulocytes / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism
D000199:Actins; D006021:Glycophorins; D013049:Spectrin