Compositional characterization of the cytoskeleton of NK-like cells.
The cytoskeleton is a dynamic structure that contributes to cell function in terms of shape, movement, transport and secretion. It also provides a platform for regional activities such as signaling, biosynthesis and energy production. The present manuscript describes a method for cytoskeleton isolation based on capture with magnetic microbeads and its application to the analysis of the NK like cell line, YTS. The isolated proteins were separated by SDS-PAGE and the peptides from the in gel digested proteins were analyzed by on line nano-LC-MSMS. Approximately 76% of the 126 isolated proteins were either components of the cytoskeleton or proteins that were known to be capable of associating with the cytoskeleton. The enrichment was confirmed by western blot for actin and alpha-actinin. The isolation was dependent on intact actin microfilaments as pretreatment of cells with cytochalasin D resulted in a marked reduction in the number of proteins isolated. The method allowed for the identification of several proteins that have not been previously described in lymphoid cells (EPLIN, SETA). A number of other scaffolding and lipid raft associated proteins were described suggesting a link between the cytoskeleton and these structures. The approach may have application to the proteomic examination of the cytoskeleton in a variety of cell types.
Meng
Xiaobo
X
Manitoba Centre for Proteomics and Systems Biology, Rheumatic Diseases Research Laboratory, Department of Internal Medicine, University of Manitoba, Canada.
Wilkins
John A
JA
D008841:Actin Cytoskeleton; D000199:Actins / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002853:Chromatography, Liquid; D015638:Cytochalasin D / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology*; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry; D008853:Microscopy; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*
D000199:Actins; D008055:Lipids; D010455:Peptides; D020543:Proteome; D015638:Cytochalasin D