Caspase Remodeling of the Spectrin Membrane Skeleton during Lens Development and Aging

Abstract

Terminal differentiation of lens fiber cells resembles the apoptotic process in that organelles are lost, DNA is fragmented, and changes in membrane morphology occur. However, unlike classically apoptotic cells, which are disintegrated by membrane blebbing and vesiculation, aging lens fiber cells are compressed into the center of the lens, where they undergo cell-cell fusion and the formation of specialized membrane interdigitations. In classically apoptotic cells, caspase cleavage of the cytoskeletal protein ␣-spectrin to ϳ150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave ␣-spectrin to ϳ150-kDa fragments and ␤-spectrin to ϳ120-and ϳ80-kDa fragments during late embryonic chick lens development. These fragments continue to accumulate with age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is cleaved to discrete fragments. Thus, unlike classical apoptosis, where caspase-cleaved spectrin is short lived, lens fiber cells contain spectrin fragments that appear to be stable for the lifetime of the organism. Moreover, fragmentation of spectrin results in reduced membrane association and thus may lead to permanent remodeling of the membrane skeleton. Partial and specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.The spectrin-actin membrane skeleton underlies the plasma membranes of all cells and is important for cellular shape, membrane stability and deformability, as well as the formation of membrane subdomains (1). The major component of the membrane skeleton, spectrin, is composed of an ␣/␤ heterodimer that self-associates head-to-head to form a 200-nm extended tetramer filament. Spectrin cross-links actin filaments into an isotropic meshwork. This spectrin-actin meshwork is attached to the membrane by direct interactions of ␤-spectrin with membrane proteins and indirect interactions of ␤-spectrin with membrane attachment proteins such as ankyrin (2).Proteolysis of ␣-spectrin (␣II-spectrin, non-erythroid spectrin, or fodrin) to discrete fragments is implicated in changes in cell shape and membrane morphology which occur in many cell types. During platelet activation, which includes a cell shape transformation from discs into irregular spheres, spectrin is cleaved to ϳ150-kDa fragments by the calcium-dependent protease, calpain (3). ␣-Spectrin cleavage by calpain has also been implicated in cellular hypoxia (4), neuronal injury and degeneration (5), and neuronal growth cone formation (6). However, in apoptotic cells, ␣-spectrin proteolysis to ϳ150-kDa fragments is mediated by caspases; in these cells, spectrin proteolysis is thought to be important for the disintegration of the plasma membranes via formation of vesicular "apoptotic bodies" (7-12). Although calpain cleavage of spectrin is known to affect its ability to bind membranes or actin filaments (13, 14), the detailed consequences of caspase cleavage of spectrin have not been studied.The terminal ...

Sequential Degradation of αII and βII Spectrin by Calpain in Glutamate or Maitotoxin-Stimulated Cells

“…8), established that this peptide is cleaved at a site identical to that of the native αII spectrin heterotetramer (Table I). Calpain did not appreciably digest peptides GST-αII [13][14][15][16][17][18] and GST-αII 18-C (data not shown). Calpain digestion of GST-βII 8-CΔ , a fusion peptide encompassing βII spectrin repeat units 8 to 17 and a portion of domain III (58), yielded four cleavage fragments at ≈ 92, ≈ 88, ≈ 70, and ≈ 47 kD (Figure 3).…”

Autonomous functions for the Sec14p/spectrin-repeat region of Kalirin

“…In many spectrin family members, the rigid structure of the spectrin repeat regions ensures proper spacing between N-and C-terminal functional groups [28,29]. The nine spectrin repeats of Kal7 separate the Sec14p domain, which can bind phosphatidylinositol (3,5)P 2 , from both the GEF domain and the PDZ binding motif.…”

Role of the Executioner Caspases during Lens Development

“…Differentiating lens fiber cells express lamin A/C and lamin B, and both are degraded during organelle breakdown (15,20). Spectrin, an actin-cross-linking protein, is also a caspase-3 substrate and is cleaved at the onset of organelle loss (21). Furthermore, the treatment of lentoid cultures (small clusters of lens cells expressing fiber cell differentiation markers) with the pancaspase inhibitors t-butoxycarbonyl-Asp-FMK or benzyloxycarbonyl-VAD-FMK, prevents the cleavage of poly(ADP-ribose) polymerase and reduces the number of TUNEL-positive nuclei in the cultures (19,22).…”