Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.
Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)
Fowler
V M
VM
Department of Cell and Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Adam
E J
EJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007399:Interphase / Q000502:physiology; D008565:Membrane Proteins / Q000502:physiology; D008938:Mitosis / Q000502:physiology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D012995:Solubility; D013049:Spectrin / Q000032:analysis / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry
D000596:Amino Acids; D008565:Membrane Proteins; D010748:Phosphopeptides; D013049:Spectrin; D011092:Polyethylene Glycols