Caspase Remodeling of the Spectrin Membrane Skeleton during Lens Development and Aging
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m009723200
ISSN1083-351X
AutoresAndria Lee, Jon S. Morrow, Velia M. Fowler,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoTerminal 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 α-spectrin to ∼150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave α-spectrin to ∼150-kDa fragments and β-spectrin to ∼120- and ∼80-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.AF354639 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 α-spectrin to ∼150-kDa fragments is believed to be important for membrane blebbing. We report that caspase(s) cleave α-spectrin to ∼150-kDa fragments and β-spectrin to ∼120- and ∼80-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.AF354639 3-(cyclohexylamino)propanesulfonic acid membrane association domain supernatant 1 and supernatant 2, respectively 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 (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar). The major component of the membrane skeleton, spectrin, is composed of an α/β 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 β-spectrin with membrane proteins and indirect interactions of β-spectrin with membrane attachment proteins such as ankyrin (2Lambert S. Bennett V. Eur. J. Biochem. 1993; 211: 1-6Crossref PubMed Scopus (51) Google Scholar).Proteolysis of α-spectrin (αII-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 ∼150-kDa fragments by the calcium-dependent protease, calpain (3Fox J.E.B. Reynolds C.C. Morrow J.S. Phillips D.R. Blood. 1987; 69: 537-545Crossref PubMed Google Scholar). α-Spectrin cleavage by calpain has also been implicated in cellular hypoxia (4Glantz S. Morrow J. Haddad G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular, and Integrated Function. Marcel Dekker, New York1995: 153-191Google Scholar), neuronal injury and degeneration (5Roberts-Lewis J.M. Siman R. Ann. N. Y. Acad. Sci. 1993; 679: 78-86Crossref PubMed Scopus (49) Google Scholar), and neuronal growth cone formation (6Gitler D. Spira M.E. Neuron. 1998; 20: 1123-1135Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, in apoptotic cells, α-spectrin proteolysis to ∼150-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" (7Wang K.K. Posmantur R. Nath R. McGinnis K. Whitton M. Talanian R.V. Glantz S.B. Morrow J.S. J. Biol. Chem. 1998; 273: 22490-22497Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 8Pike B.R. Zhao X. Newcomb J.K. Wang K.K. Posmantur R.M. Hayes R.L. J. Neurosci. Res. 1998; 52: 505-520Crossref PubMed Scopus (65) Google Scholar, 9Jordan J. Galindo M.F. Miller R.J. J. Neurochem. 1997; 68: 1612-1621Crossref PubMed Scopus (170) Google Scholar, 10Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 11Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar, 12Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T.C. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Although calpain cleavage of spectrin is known to affect its ability to bind membranes or actin filaments (13Hu R.-J. Bennett V. J. Biol. Chem. 1991; 266: 18200-18205Abstract Full Text PDF PubMed Google Scholar, 14Harris A.S. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3009-3013Crossref PubMed Scopus (105) Google Scholar), the detailed consequences of caspase cleavage of spectrin have not been studied.The terminal differentiation and aging of lens fiber cells are marked by dramatic membrane morphological changes. As new cells arise on the outside of the lens, older cells are pushed inward, where organelles are lost, and cells fuse to form a syncytium (15Rafferty N. Maisel H. The Ocular Lens: Structure, Function, and Pathology. Marcel Dekker, New York1985: 1-60Google Scholar, 16Shestopalov V. Bassnett S. J. Cell Sci. 2000; 113: 1913-1921PubMed Google Scholar). Because these cells are never lost from the lens, the most central cells are as old as the organism. In addition, as lens fiber cells mature, specialized membrane interdigitations develop, which are distributed regularly on the lateral membranes (17Kuszak J.R. Peterson K.L. Brown H.G. Microsc. Res. Tech. 1996; 33: 441-479Crossref PubMed Scopus (36) Google Scholar). These age-related changes in membrane morphology have been compared with the formation of apoptotic bodies (18Dahm R. Ophthalmic Res. 1999; 31: 163-183Crossref PubMed Scopus (93) Google Scholar). In addition, they are believed to be important for lens transparency by reducing light scattering at cell boundaries and by allowing for protein turnover and ion homeostasis (16Shestopalov V. Bassnett S. J. Cell Sci. 2000; 113: 1913-1921PubMed Google Scholar).We have shown previously that spectrin and other components of the membrane skeleton are associated with the plasma membranes of young and old lens fiber cells (19Lee A. Fischer R.S. Fowler V.M. Dev. Dyn. 2000; 217: 257-270Crossref PubMed Scopus (78) Google Scholar). Here, we report that α-spectrin is cleaved to ∼150-kDa fragments during terminal differentiation and aging of lens fiber cells. In addition, β-spectrin, which dimerizes with α-spectrin, is also proteolyzed to ∼120- and ∼80-kDa fragments. Fragmentation of spectrin progresses with lens and fiber cell age so that in the oldest fiber cells of the adult lens, most, if not all, of the spectrin is fragmented. The spectrin-binding and membrane-binding protein, ankyrin, is also partially proteolyzed with lens fiber cell age. N-terminal amino acid sequencing of the spectrin fragments reveals that caspase cleavage is responsible for lens spectrin proteolysis. Moreover, subcellular fractionation of lens fiber cells indicates that caspase-cleaved spectrin fragments display reduced association with lens membranes. The specific proteolysis of membrane skeleton components by caspases may be important for age-related membrane changes in the lens.DISCUSSIONThis is the first report of caspase cleavage of membrane skeleton proteins to discrete and stable fragments during cellular maturation and aging. We have shown that α-spectrin is cleaved to ∼150-kDa fragments and β-spectrin to ∼120- and ∼80-kDa fragments during lens fiber cell maturation and aging. The spectrin-binding protein, ankyrin, is also cleaved to ∼190-kDa fragments. These fragments appear to be extremely stable and indeed, accumulate with age. In contrast, in other cell types, caspase cleavage of spectrin precedes cell death, thus ensuring a short half-life for the spectrin fragments (7Wang K.K. Posmantur R. Nath R. McGinnis K. Whitton M. Talanian R.V. Glantz S.B. Morrow J.S. J. Biol. Chem. 1998; 273: 22490-22497Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 8Pike B.R. Zhao X. Newcomb J.K. Wang K.K. Posmantur R.M. Hayes R.L. J. Neurosci. Res. 1998; 52: 505-520Crossref PubMed Scopus (65) Google Scholar, 9Jordan J. Galindo M.F. Miller R.J. J. Neurochem. 1997; 68: 1612-1621Crossref PubMed Scopus (170) Google Scholar, 10Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 11Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K. Biochem. J. 1996; 319: 683-690Crossref PubMed Scopus (398) Google Scholar, 12Martin S.J. O'Brien G.A. Nishioka W.K. McGahon A.J. Mahboubi A. Saido T.C. Green D.R. J. Biol. Chem. 1995; 270: 6425-6428Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). Moreover, when cleavage of spectrin does not lead to cell death, (i.e. neuronal remodeling), a brief period of accumulation of fragments is followed by a decrease in the proportion of fragments to full-length spectrin polypeptides, suggesting replacement of spectrin fragments with newly synthesized full-length spectrin (36Di Stasi A.M. Gallo V. Ceccarini M. Petrucci T.C. Neuron. 1991; 6: 445-454Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Our results indicate that in lens fiber cells, which do not undergo cell death, the accumulation of caspase cleavage products of spectrin and ankyrin may lead to a permanent remodeling of the membrane skeleton.The cleavage of membrane skeleton components in the lens is specific. In contrast to spectrin and ankyrin, other components of the membrane skeleton, tropomodulin, tropomyosin, and actin, do not appear to be proteolyzed during lens fiber cell aging (19Lee A. Fischer R.S. Fowler V.M. Dev. Dyn. 2000; 217: 257-270Crossref PubMed Scopus (78) Google Scholar). However, there are reports of other membrane-associated lens proteins being proteolyzed to discrete and stable fragments. Partial proteolysis of the major lens membrane protein, MP-26 from a ∼26- kDa to a ∼20-kDa protein, has been reported to occur during fiber cell maturation (37Hoenders H.J. Bloemendal H. Bloemendal H. The Molecular and Cellular Biology of the Eye Lens. John Wiley and Sons, New York1981: 279-326Google Scholar). The gap junction protein connexin 50 (α8) has been shown to be cleaved by calpain, leading to removal of the C-terminal tail of the protein from the plasma membranes (38Lin J.S. Fitzgerald S. Dong Y. Knight C. Donaldson P. Kistler J. Eur. J. Cell Biol. 1997; 73: 141-149PubMed Google Scholar). Connexin 46 (α3) may also be cleaved in mature fiber cells (39Berthoud V.M. Cook A.J. Beyer E.C. Invest. Ophthalmol. Visual Sci. 1994; 35: 4109-4117PubMed Google Scholar). The lens-specific intermediate filament protein filensin is also proteolyzed to a discrete ∼53-kDa band in maturing fiber cells in the bovine lens (40Sandilands A. Prescott A. Hutcheson A. Quinlan R. Casselman J. FitzGerald P. Eur J. Cell Biol. 1995; 67: 238-253PubMed Google Scholar) and multiple bands in the chicken lens (19Lee A. Fischer R.S. Fowler V.M. Dev. Dyn. 2000; 217: 257-270Crossref PubMed Scopus (78) Google Scholar). Finally, ∼150-kDa α-spectrin fragments have been observed in the rabbit and guinea pig lens (41Fukiage C. Azuma M. Nakamura Y. Tamada Y. Shearer T.R. Curr. Eye Res. 1998; 17: 623-635Crossref PubMed Google Scholar) in addition to our observations in the chicken, rat, and cow lens. Partial proteolysis of particular membrane-associated proteins (and not others) to discrete and stable fragments may be important for membrane remodeling during lens fiber cell aging.The timing of spectrin fragmentation also implies roles in mediating membrane morphological changes during lens fiber cell aging. Spectrin fragments are first detected late during embryonic development, coincident with formation of the organelle-free zone (28Bassnett S. Beebe D.C. Dev. Dyn. 1992; 194: 85-93Crossref PubMed Scopus (188) Google Scholar), but continue to accumulate during development and after hatching. In the adult lens, spectrin fragmentation appears to be restricted to older fiber cells, and the amount of spectrin fragmentation increases with the age of the lens fiber cell. Unfortunately, it is difficult to compare the timing of spectrin fragmentation with that of other membrane-associated proteins because a similar developmental analysis has not been performed with other proteins. However, a functional syncytium of cells within the organelle-free zone is first detected at day 12 of embryonic development and expands with age (16Shestopalov V. Bassnett S. J. Cell Sci. 2000; 113: 1913-1921PubMed Google Scholar). In addition, membrane protrusions are not present in the day 7 embryonic lens but are first observed later in the day 10 embryonic lens; these membrane protrusions become more elaborate with age (42Shestopalov V. Bassnett S. Invest. Ophthalmol. Visual Sci. 2000; 41: 859-863PubMed Google Scholar). Moreover, in the adult chicken lens, newly differentiated cortical fiber cells exhibit smooth profiles, whereas older fiber cells display numerous membrane protrusions (43Kuszak J. Alcala J. Maisel H. Am. J. Anat. 1980; 159: 395-410Crossref PubMed Scopus (63) Google Scholar). In support of a role for spectrin fragmentation in the development of membrane protrusions, antibodies, which recognize both full-length and fragments of α-spectrin stain protrusions of nuclear fiber cells (19Lee A. Fischer R.S. Fowler V.M. Dev. Dyn. 2000; 217: 257-270Crossref PubMed Scopus (78) Google Scholar) as well as blebs decorating differentiated lens cells in culture. 2B. Fischer and V. M. Fowler, unpublished observations.This is the first report of caspase-mediated cleavage of membrane-associated components in the lens. Our data suggests that caspase-3 may be involved in proteolysis of spectrin during lens fiber cell maturation and aging. The sequence of the cleavage site for chicken lens α-spectrin is identical to that obtained by in vitro cleavage of bovine brain αII-spectrin by caspase-3. In addition, the sequences of cleavage for both chicken lens α- and β-spectrins (DETD*S, DEVD*S) match well with the consensus DXXD*S cleavage site identified for other caspase-3 substrates (44Earnshaw W. Martins L. Kaufmann S. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2429) Google Scholar). In rat and cow lenses, the presence of the ∼110–120-kDa α-spectrin fragment generated by caspase-3 cleavagein vitro also suggests cleavage of endogenous lens α-spectrin by caspase-3 (7Wang K.K. Posmantur R. Nath R. McGinnis K. Whitton M. Talanian R.V. Glantz S.B. Morrow J.S. J. Biol. Chem. 1998; 273: 22490-22497Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 45Wang K. Trends Neurosci. 2000; 23: 20-26Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). The lack of a ∼120-kDa fragment of α-spectrin in the chicken lens may be due to conformational differences between chicken and mammalian spectrins.Caspase activity has previously been shown to be necessary for loss of nuclei during lens fiber cell differentiation (46Wride M.A. Parker E. Sanders E.J. Dev. Biol. 1999; 213: 142-156Crossref PubMed Scopus (76) Google Scholar, 47Ishizaki Y. Jacobson M.D. Raff M.C. J. Cell Biol. 1998; 140: 153-158Crossref PubMed Scopus (257) Google Scholar, 48Kuida K. Zheng T. Na S. Kuan C.-y. Yang D. Harasuyama H. Rakic P. Flavell R. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1700) Google Scholar). In addition, members of the caspase family (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar, 2Lambert S. Bennett V. Eur. J. Biochem. 1993; 211: 1-6Crossref PubMed Scopus (51) Google Scholar, 3Fox J.E.B. Reynolds C.C. Morrow J.S. Phillips D.R. Blood. 1987; 69: 537-545Crossref PubMed Google Scholar, 4Glantz S. Morrow J. Haddad G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular, and Integrated Function. Marcel Dekker, New York1995: 153-191Google Scholar, 6Gitler D. Spira M.E. Neuron. 1998; 20: 1123-1135Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and the caspase substrates, DNA fragmentation factor and poly(ADP-ribose) polymerase, have been identified in the lens (46Wride M.A. Parker E. Sanders E.J. Dev. Biol. 1999; 213: 142-156Crossref PubMed Scopus (76) Google Scholar). Like spectrin, poly(ADP-ribose polymerase has also been reported to be cleaved late in lens development, after organelle loss (46Wride M.A. Parker E. Sanders E.J. Dev. Biol. 1999; 213: 142-156Crossref PubMed Scopus (76) Google Scholar).Although our data suggest that caspase cleavage of the membrane skeleton occurs after organelle loss, others have reported that caspase activation is required for organelle loss. This apparent discrepancy may be explained by the idea that different caspases may be active at different times during lens fiber cell maturation. According to Wrideet al. (46Wride M.A. Parker E. Sanders E.J. Dev. Biol. 1999; 213: 142-156Crossref PubMed Scopus (76) Google Scholar), particular caspases (i.e. caspase-3) are present early during lens fiber cell differentiation, whereas others (i.e. caspase-1) are predominant in older lens fiber cells. Moreover, although inhibitors of caspases-1, -2, -6, and -9 appeared to inhibit nuclear loss in lens cell cultures, inhibitors of caspase-3 and -8 were ineffective. Both of these observations suggest that although the caspase pathway has not been delineated precisely for lens cells, particular caspases are important at different times during lens fiber cell differentiation. Thus, it is possible that specific caspases (particularly caspase-3) are important for selective proteolysis of the membrane skeleton after organelle loss, whereas other caspases are important for initiating organelle loss.What are the molecular consequences of caspase cleavage of spectrin and ankyrin? We have shown that spectrin fragments are partially dissociated from lens membranes. This may be due to cleavage of ankyrin, which binds to both β-spectrin and integral membrane proteins. Calpain-cleaved ankyrin, which, like lens ankyrin, is also a ∼190-kDa fragment derived from cleavage near the C terminus, exhibits an 8-fold weaker affinity for erythrocyte membranes (49Hall T. Bennett V. J. Biol. Chem. 1987; 262: 10537-10545Abstract Full Text PDF PubMed Google Scholar). Alternatively, cleavage of β-spectrin could lead to reduced membrane association. However, the initial site of cleavage near the middle of the protein is not within its ankyrin binding site, actin binding site, or within any of the ankyrin-independent MADs (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar). Thus, β-spectrin cleavage is unlikely to interfere with membrane association by direct interference with its membrane binding sites, but perhaps indirectly by affecting the conformation of these sites.Cleavage of lens spectrin by caspase may lead to lowered membrane affinity as well as calmodulin down-regulation of spectrin activities. The caspase cleavage site on α-spectrin is within 9 amino acids of the calpain cleavage site and is proximal to the calmodulin binding site (Fig. 4 B). When calmodulin is bound to α-spectrin during the action of calpain, both α- and β-spectrin subunits are cleaved, and the heterotetramer dissociates irreversibly into its component fragments (14Harris A.S. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3009-3013Crossref PubMed Scopus (105) Google Scholar). This results in complete loss of tetramer formation, F-actin binding, or membrane binding. Such a dissociation of spectrin fragments is reminiscent of the dissociation reported here which results from caspase-mediated cleavage of both subunits in the intact heterodimer.Interestingly, α- and β-spectrin are both cleaved at repeat 11, at sites that interact in the α-β-spectrin heterodimer (50Viel A. Branton D. Curr. Opin. Cell Biol. 1996; 8: 49-55Crossref PubMed Scopus (66) Google Scholar), suggesting that spectrin may be fully assembled before cleavage. In contrast, in other types of cells (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar), calpain may either cleave the α- or β-spectrin subunits separately or together in the intact heterotetramer, thus leading to targeted loss of tetramer assembly, F-actin binding, or membrane binding (13Hu R.-J. Bennett V. J. Biol. Chem. 1991; 266: 18200-18205Abstract Full Text PDF PubMed Google Scholar, 14Harris A.S. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3009-3013Crossref PubMed Scopus (105) Google Scholar). Thus, although other cell types may use a multistep process involving calpain and calmodulin to accomplish an incrementally regulated disassembly of the spectrin cortical skeleton, lens cells may use predominately caspase and calmodulin to disassemble their membrane skeleton in a single step process.We have developed a speculative model (Fig.6) to describe how specific and partial proteolysis of membrane skeleton proteins could lead to membrane morphological changes in the lens. In young (cortical) fiber cells (Fig. 6 A), as in other non-erythroid cells, α-β-spectrin tetramers are likely cross-linked to short actin filaments. This two-dimensional meshwork is then anchored to the membranes via spectrin interactions with ankyrin and other membrane proteins (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar, 51Bennett V. Physiol. Rev. 1990; 70: 1029-1065Crossref PubMed Scopus (356) Google Scholar). In older fiber cells (Fig. 6 B), localized caspase activity could result in proteolytic cleavage of specific regions of the membrane skeleton, loosening constraints on the membranes and thus allowing for membrane blebbing in specific sites. The extent and placement of membrane blebbing could be regulated by whether ankyrin, α-spectrin, and/or β-spectrin are cleaved, or by calmodulin binding. With age, more caspase activity, and thus, more proteolytic processing, would occur. This could result in the increased density of membrane protrusions with fiber cell age (17Kuszak J.R. Peterson K.L. Brown H.G. Microsc. Res. Tech. 1996; 33: 441-479Crossref PubMed Scopus (36) Google Scholar). Membrane blebbing could lead to repositioning of integral membrane proteins, such as ion channels, which might be necessary for age-related changes in ion and water flow (52Mathias R.T. Rae J.L. Baldo G.J. Physiol. Rev. 1997; 77: 21-50Crossref PubMed Scopus (339) Google Scholar). Moreover, membrane blebbing might lead to cell-cell fusion events that have been observed in maturing lenses (16Shestopalov V. Bassnett S. J. Cell Sci. 2000; 113: 1913-1921PubMed Google Scholar, 17Kuszak J.R. Peterson K.L. Brown H.G. Microsc. Res. Tech. 1996; 33: 441-479Crossref PubMed Scopus (36) Google Scholar).Interestingly, cleavage of α-spectrin by calpain, not by caspase, has been associated with a number of cataract models (41Fukiage C. Azuma M. Nakamura Y. Tamada Y. Shearer T.R. Curr. Eye Res. 1998; 17: 623-635Crossref PubMed Google Scholar, 53Kilic F. Trevithick J.R. Biochem. Mol. Biol. Int. 1998; 45: 963-978PubMed Google Scholar). Inhibition of calpain inhibits cataract formation and spectrin cleavage (54Wang K.K. Yuen P.W. Adv. Pharmacol. 1997; 37: 117-152Crossref PubMed Scopus (94) Google Scholar), and human lenses with age-related nuclear cataracts display a higher density of finger-like membrane projections than transparent lenses of the same age (55Boyle D.L. Takemoto L.J. Curr. Eye Res. 1998; 17: 1118-1123Crossref PubMed Scopus (6) Google Scholar). Thus, it is possible that aberrant cleavage of spectrin by calpain may lead to uncontrolled or incomplete membrane furrowing and opacification of the lens, whereas specific cleavage of spectrin by caspase may be important for normal physiological functioning of the lens.In conclusion, partial and specific proteolysis of spectrin and ankyrin by caspases appear to effect an apoptosis-like program of membrane changes during lens aging. Further characterization of lens membrane morphology and membrane skeleton proteolysis in caspase knockouts, as well as additional biochemical analysis of the lens membrane skeleton, will lead to greater insight in the importance of caspase cleavage of membrane skeleton components during lens development and function. It is likely that the transparency of the aging lens nucleus may depend not only on organelle loss but also on membrane skeleton remodeling. We anticipate that these changes in the membrane skeleton, induced by caspase-mediated but limited proteolysis, will modulate ion homeostasis, the positioning of ion channels, the frequency of intercellular fusion events, and lens deformability. 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 (1Morrow J. Rimm D. Kennedy S. Cianci C. Sinard J. Weed S. Hoffman J. Jamieson J. Handbook of Physiology. Oxford University Press, London1997: 485-540Google Scholar). The major component of the membrane skeleton, spectrin, is composed of an α/β 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 β-spectrin with membrane proteins and indirect interactions of β-spectrin with membrane attachment proteins such as ankyrin (2Lambert S. Bennett V. Eur. J. Biochem. 1993; 211: 1-6Crossref PubMed Scopus (51) Google Scholar). Proteolysis of α-spectrin (αII-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 ∼150-kDa fragments by the calcium-dependent protease, calpain (3Fox J.E.B. Reynolds C.C. Morrow J.S. Phillips D.R. Blood. 1987; 69: 537-545Crossref PubMed Google Scholar). α-Spectrin cleavage by calpain has also been implicated in cellular hypoxia (4Glantz S. Morrow J. Haddad G. Lister G. Tissue Oxygen Deprivation: Developmental, Molecular, and Integrated Function. Marcel Dekker, New York1995: 153-191Google Scholar), neuronal injury and degeneration (5Roberts-Lewis J.M. Siman R. Ann. N. Y. Acad. Sci. 1993; 679: 78-86Crossref PubMed Scopus (49) Google Scholar), and neuronal growth cone formation (6Gitler D. Spira M.E. Neuron. 1998; 20: 1123-1135Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, in apoptotic cells, α-spectrin proteolysis to ∼150-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" (7Wang K.K. Posmantur R. Nath R. McGinnis K. Whitton M. Talanian R.V. Glantz S.B. Morrow J.S. J. Biol. Chem. 1998; 273: 22490-22497Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 8Pike B.R. Zhao X. Newcomb J.K. Wang K.K. Posmantur R.M. Hayes R.L. J. Neurosci. Res. 1998; 52: 505-520Crossref PubMed Scopus (65) Google Scholar, 9Jordan J. Galindo M.F. Miller R.J. J. Neurochem. 1997; 68: 1612-1621Crossref PubMed Scopus (170) Google Scholar, 10Cryns V.L. Bergeron L. Zhu H. Li H. Yuan J. J. Biol. Chem. 1996; 271: 31277-31282Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 11Nath R. Raser K.J. Stafford D. Hajimohammadreza I. Posner A. Allen H. Talanian R.V. Yuen P. Gilbertsen R.B. Wang K.K. Bioche
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