In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s).

Abstract. 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 cellsubstratum 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 $3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25 %).The fl 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 fl 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 mitosisspecific phosphorylation of fl-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. We propose that mitosis-specific redistribution of spectrin to the cytosol is a consequence of phosphorylation by mitotic kinases and reflects spectrin dissociation from the plasma membrane. This could be functionally related to restructuring of membraneassociated actin filament networks in mitotic cells and could play ...

Section: Spectrin Solubility In Interphase and Mitotic Cho Cellsmentioning

confidence: 77%

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells.

Fowler

Adam

1992

“…The reorganization of the membrane skeleton in mdx myofibers may be an indirect result of the absence of dystrophin, perhaps related to changes in Ca 2ϩ homeostasis (21,33,36,44,66,78). Ca 2ϩ -dependent proteases, such as calpain and caspases, activated by Ca 2ϩ entering the myofiber through the dystrophic sarcolemma, are capable of cleaving spectrins (34,61,67,81) and other structural proteins (29). The proteolysis of spectrin, coupled with the absence of dystrophin, might disrupt the membrane-associated cytoskeleton sufficiently to destabilize sarcolemmal organization.…”

Section: Discussionmentioning

confidence: 99%

Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice

Williams

Bloch

1999

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. β-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, β-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, β-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.

“…Spectrin-bound annexin VI could regulate the remodeling of membrane spectrin by activating a cytosolic protease. Since calpain I has been implicated in removing spectrin from the membrane (31,34), we used the cysteine protease inhibitor ALLN to see if it would block annexin VI-dependent removal of spectrin (Fig. 4 A).…”

Section: Annexin VI Mediates Remodeling Of Membrane Cytoskeletonmentioning

confidence: 99%

“…The removal of spectrin may be mediated by the cysteine protease calpain I. Calpain I cleaves both the ␣ and ␤ subunits of brain spectrin, producing subunits that are no longer able to form tetramers or interact with actin (31). Limited proteolysis by calpain also inhibits spectrin from associating with membrane-binding sites (34).…”

mentioning

confidence: 99%

Annexin VI-mediated Loss of Spectrin during Coated Pit Budding Is Coupled to Delivery of LDL to Lysosomes

Kamal

Ying

Anderson

1998

100

Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI–dependent budding is accompanied by the loss of ∼50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI–dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.