Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines.
Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.
Damaraju
Sambasivarao
S
PolyomX Program, Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada T6G 1Z2.
Zhang
Nancy
N
Li
Nan
N
Tao
Lidan
L
Damaraju
Vijaya L
VL
Dufour
Jennifer
J
Santos
Cheryl
C
Sun
Xue-Jun
XJ
Mackey
John
J
Wishart
David S
DS
Cass
Carol E
CE
Li
Liang
L
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004273:DNA, Neoplasm / Q000032:analysis; D006801:Humans; D051379:Mice; D048629:Micronuclei, Chromosome-Defective; D009687:Nuclear Proteins / Q000032:analysis; D013347:Subcellular Fractions / Q000201:enzymology
D015415:Biomarkers; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D000469:Alkaline Phosphatase