Correspondence to Michael Olson: m.olson{at}beatson.gla.ac.uk
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations used in this paper: 4-HT, 4-hydroxytamoxifen; CHX, cycloheximide; LAP, lamin-associated protein; LIMK, LIM kinase; MEF, mouse embryo fibroblast; MLC, myosin light chain; PARP, poly-ADP ribose polymerase; TEM, transmission EM.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dynamic contraction and membrane blebbing seen in apoptotic cells are dependent on intracellular force generated by the actin-myosin cytoskeleton. These morphological events are controlled by the Rho effector ROCK I, a serine/threonine kinase that plays a key and central role in the regulation of actin cytoskeletal structures. We and others showed that caspase-mediated cleavage of ROCK I results in constitutive activation and consequent myosin light chain (MLC) phosphorylation leading to contraction and membrane blebbing (Coleman et al., 2001; Sebbagh et al., 2001). Inhibition of ROCK activity with the small molecule inhibitor Y-27632 attenuated blebbing in a variety of cell types, independent of the type of apoptotic stimulus. Inhibition of ROCK activity also prevented the relocalization of fragmented DNA into membrane blebs and apoptotic bodies (Coleman et al., 2001), suggesting additional roles for ROCK in the morphological changes that occur during apoptosis.
In addition to the gross external morphological responses, there are significant effects on the morphology and integrity of organelles, the most obvious being nuclear disintegration. Separating the nucleus from the cytoplasm is the nuclear envelope, which is comprised of outer and inner nuclear membranes. Giving the nucleus form, structure, and rigidity is a filamentous meshwork called the lamina, which is made up from intermediate filament A-type (A and C, alternately spliced products from the Lmna gene) and B-type (B1, B2, and B3) lamins. Caspase-mediated cleavage of lamins A/C and B1 is thought to contribute to nuclear fragmentation during apoptosis (Neamati et al., 1995; Rao et al., 1996; Broers et al., 2002).
Ultrastructural analysis has shown that the nucleus is surrounded by a meshwork of actin (Clubb and Locke, 1998b), with "knots" of actin physically associated with the nuclear envelope (Clubb and Locke, 1998a). Disruption of the actin cytoskeleton alters nuclear morphology (Zhen et al., 2002), while mutations to Anc-1/Syne family actin-binding proteins result in aberrant nuclear anchoring (Starr and Han, 2003), indicating that the actin cytoskeleton influences nuclear positioning, shape, and structure. Therefore, one possibility is that during apoptosis, active caspase-cleaved ROCK I leads to shortening of actin-myosin filaments that are tethered to the nucleus at one end, resulting in nuclear envelope tearing and disintegration, thereby allowing for the relocalization of fragmented DNA to membrane blebs and apoptotic bodies (Coleman et al., 2001). Mitotic nuclear envelope breakdown also requires weakening of the nuclear lamina and a pulling force, but is mediated by phosphorylation-induced depolymerization of the nuclear lamina (Heald and McKeon, 1990) and microtubule-anchored pulling force generated by the minus-enddirected motor, cytoplasmic dynein, and components of its associated regulatory complex, dynactin (Beaudouin et al., 2002; Salina et al., 2002).
In this work, we examined the contribution of ROCK activity and MLC phosphorylation to nuclear disintegration during apoptosis. We found that ROCK activity, intact actin filaments, MLC phosphorylation, and MLC ATPase activity are each required for the breakdown of nuclear structure, whereas intact microtubules are dispensable. Caspase-mediated cleavage of lamins A/C and B1 is not sufficient for nuclear disintegration in the absence of ROCK and MLC ATPase activity. In addition, conditional activation of ROCK I induces nuclear breakdown in nonapoptotic cells only in the absence of lamin A/C expression. These results indicate that apoptotic nuclear breakdown requires weakening of the nuclear lamina by proteolytic cleavage and the contractile force generated by ROCK on actin-myosin filaments. Thus, apoptotic nuclear breakdown parallels mitotic nuclear breakdown in the requirements for lamina disassembly and generation of pulling force, but differs in the means by which these events are achieved.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We used transmission EM (TEM) to obtain fine-detailed photomicrographs of nuclear morphology during apoptosis. NIH 3T3 mouse fibroblasts were treated with 25 ng/ml TNF plus 10 µg/ml cycloheximide (CHX) for 2 h as the apoptotic stimulus, and then processed for TEM. A typical apoptotic cell is shown in Fig. 1 A, with membrane blebs, chromatin condensation, and a significantly fragmented nucleus with large invaginations of the nuclear envelope and evidence of electron-dense heterochromatin within the cytoplasm and in blebs (Fig. 1 A, arrows). In contrast, cells given the ROCK inhibitor Y-27632 (10 µM) in conjunction with TNF
/CHX did not bleb and the nucleus remained intact, although chromatin condensation and nuclear envelope dilation were evident (Fig. 1 B). Consistent with our previous observations (Coleman et al., 2001), heterochromatin was retained within the nucleus, indicating that the nuclear envelope remained a barrier to macromolecules.
|
Additional downstream substrates of ROCK that may contribute to actin-myosin contractility are the LIM kinases (LIMK) 1 and 2 (Ohashi et al., 2000; Sumi et al., 2001). Overexpression of LIMK2 has been reported to induce the formation of stress fibers and membrane blebs (Amano et al., 2001), suggesting that LIMK1/2 activation contributes to the apoptotic morphology. Using an antibody that detects phosphorylation of a Thr residue in the conserved activation loops of LIMK1 (Thr508) and LIMK2 (Thr505), we observed increased phosphorylation following the induction of apoptosis, which could be blocked with ROCK inhibitor Y-27632 or by inhibition of ROCK I cleavage with z-VAD-fmk (Fig. 1 F). These results indicate that LIMK activation may contribute to ROCK-mediated morphological effects in apoptotic cells.
We also examined how the induction of apoptosis affected the phosphorylation state of Ezrin, which acts as a linker between actin filaments and transmembrane proteins such as CD44, thereby providing an anchoring point in the plasma membrane for actin-myosin fibers. ROCK-induced contraction of NIH 3T3 cells was reported to be dependent on phosphorylation of Ezrin Thr567 (Tran Quang et al., 2000), which allows for dissociation of inter- and/or intramolecular interactions and association with actin filaments (Gautreau et al., 2000). Using an antibody specific for Ezrin phosphorylated on Thr567, no changes in the phosphorylation status were observed in apoptotic cells or after Y-27632 treatment (Fig. 1 G), indicating that ROCK-mediated phosphorylation of Ezrin on Thr567 does not contribute to apoptotic cell contraction. Treatment of cells with the general kinase inhibitor staurosporine (0.2 µM) for 30 min eliminated Ezrin T567 phosphorylation, suggesting that kinases other than ROCK are responsible for basal phosphorylation.
Nuclear disintegration and the actin and microtubule cytoskeletons
Next, we determined whether or not, in addition to ROCK activity, an intact actin cytoskeleton was required for nuclear disruption. Pretreatment with the actin-destabilizing compound cytochalasin D (2 µM; Sampath and Pollard, 1991) for 2 h before treatment with TNF/CHX disrupted the actin cytoskeleton (Fig. 2 A) and blocked fragmentation of the nucleus (Fig. 2 B). Similar results (unpublished data) were obtained when the actin cytoskeleton was disrupted with swinholide A (0.1 µM; Bubb et al., 1995) or latrunculin B (0.5 µM; Spector et al., 1983). These results indicate that an intact actin cytoskeleton is necessary to mediate nuclear disintegration. Disruption of the cytoskeleton with cytochalasin D did not affect caspase 3 activation (Fig. 2 C) and cleavage of ROCK I (Fig. 2 D) or poly-ADP ribose polymerase (PARP; Fig. 2 E), which is consistent with the lack of effect of Y-27632 on caspase activation (Fig. 1, C and E).
|
|
|
|
|
|
|
Cell contraction, blebbing, and nuclear disruption in lamin A/C null MEFs occurred after 4-HTinduced activation of ROCK I:ER in the presence of caspase inhibitor z-VAD-fmk, which is consistent with these effects being directly induced and not secondarily through the induction of apoptosis. Western blotting of lysates prepared from ROCK I:ER- and KD:ER-expressing wild-type and lamin A/C null fibroblasts revealed no evidence of lamin B1 (a caspase 6 substrate; Fig. 9 A) or PARP (a caspase 3 substrate; Fig. 9 B) cleavage, which were readily detectable in cells made apoptotic by TNF/CHX treatment. Western blotting for lamin A/C confirmed the absence of expression in the lamin A/C null cells (not depicted), whereas blotting for ß-tubulin and ERK2 showed protein loading (Fig. 9, A and C). These results reinforce the conclusion that ROCK I:ER-mediated contraction is sufficient to induce nuclear disruption in nuclei with compromised lamina structures independent of caspase activation.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One unresolved question is the identity of the protein or proteins that couple actin-myosin filaments to the nuclear envelope, thereby facilitating the disruption of nuclear integrity during apoptosis. The leading candidates for this function are the spectrin family proteins Syne-1 and Syne-2 (of which there are numerous alternative splice variants and names including Myne-1/Nesprin-1/Enaptin/CPG2 and Myne-2/Nesprin-2/NUANCE) that have been shown to associate with actin filaments at their amino-terminal ends (Zhang et al., 2001, 2002; Zhen et al., 2002; Padmakumar et al., 2004) and insert into the nuclear membrane at the carboxyl-terminal ends (Apel et al., 2000; Zhang et al., 2001; Mislow et al., 2002b; Zhen et al., 2002; Padmakumar et al., 2004), where they may directly bind A-type lamins (Mislow et al., 2002a,b). Previous research has demonstrated that disruption of the Syne homologue ANC-1 in Caenorhabditis elegans leads to defects in nuclear tethering, which result in aberrant spacing of nuclei in multinucleated syncytial cells (Starr and Han, 2002). Therefore, we propose that during apoptosis the Syne proteins contribute to nuclear disintegration by serving as the physical link that couples the contraction of actin-myosin filaments to the physical tearing of the weakened nuclear lamina and membrane. Efforts in our laboratory are currently underway to test this proposal.
Data from the lmna knockout mouse (Sullivan et al., 1999) and from RNAi studies (Harborth et al., 2001) have shown that Lamin A/C is not essential for viability. However, data from the knockout mouse and from human laminopathies (Burke and Stewart, 2002) indicate that Lamin A/C function is critical in specific tissues, most notably in skeletal and cardiac muscle where actin-myosin contractile forces are considerable. Therefore, we speculate that the viability of cells with mutated or deleted lmna is compromised in these tissues because actin-myosin contractile forces are sufficient to disrupt nuclear integrity, leading to cell death. Other tissues are apparently unaffected, likely due to actin-myosin contractile force being insufficient to significantly disrupt nuclear integrity.
Certain autoimmune diseases such as systemic lupus erythematosus are believed to arise due to immunological responses to nuclear autoantigens released from apoptotic cells. Treatment with ROCK inhibitor Y-27632 reduced the translocation of the 681 (Shiratsuchi et al., 2003) and D56R/S76R (Cocca et al., 2002) nuclear autoantigens to plasma membrane blebs and apoptotic bodies without affecting phosphatidylserine externalization (Coleman et al., 2001). The results of this work indicate that ROCK activity is required for actin-myosin contractile force generation and consequent disruption of nuclear integrity, which is required for the movement of DNA and proteins from the nucleus into blebs and apoptotic bodies. Therefore, ROCK inhibition may have some prophylactic and/or ameliorative benefits for the treatment of specific autoimmune diseases.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rat MLC cDNA (provided by L. Machesky, The University of Birmingham, Edgbaston, Birmingham, UK) was subcloned into the EcoRI restriction site of pEGFP-N3 (CLONTECH Laboratories, Inc.). Mutation of Thr18 Ser19 sites to Ala was done using a QuikChange site-directed mutagenesis kit (Stratagene) according to instructions. Human ROCK I:ER was made by subcloning a blunt-ended PCR product encoding amino acids 2537 into filled-in ScaIEcoRI sites of pBABE puro EGFP:Raf-1:ER (Woods et al., 1997; provided by M. McMahon, University of California San Francisco Comprehensive Cancer Center, San Francisco, CA). Mutation of Lys105 to Gly was done with QuikChange.
BOSC 293 ecotropic retroviral packaging cells were transfected with pBABE puro ROCK I:ER or KD:ER plasmid using Effectene (QIAGEN) according to instructions. After 36 h, supernatant was collected, centrifuged at 14,000 rpm for 15 min, and aliquots were stored at 80°C. Exponentially growing NIH 3T3 or MEFs were infected with undiluted retroviral supernatant mixed with 4 µg/ml polybrene (Sigma-Aldrich) and selected with 2.5 µg/ml puromycin (Sigma-Aldrich) to establish transduced pools.
Cytochalasin D, 4-HT, and swinholide A were from purchased from Sigma-Aldrich, z-VAD-fmk and staurosporine were purchased from BIOMOL Research Laboratories, Inc., and Y-27632 and Latrunculin B were purchased from Calbiochem.
TEM
Cell suspensions were pelleted by centrifugation, resuspended in 1 ml of fixative in an Eppendorf tube and immediately spun down again to form a pellet, and stored overnight at 4°C. Samples were postfixed in 2% osmium tetroxide, dehydrated through a graded series of ethanols, infiltrated, and embedded in Epon. For light microscopy, 1.0-µm sections were cut, dried onto microscope slides, and stained with toluidine blue. For EM, 75-nm sections were collected onto copper grids, double stained with uranyl acetate and bismuth subnitrite, and examined in a transmission electron microscope (model 1010 Jem; JEOL) at 80 Kv accelerating voltage. Digital images were collected using a CCD camera (model 2k; Hamamatsu) aided with AMT 12-HR software.
Western blot analysis
After treatment as described in the figure legends, cells were lysed in 1x Laemmli sample buffer, sonicated, and electrophoresed on 10% SDS-PAGE before electro-transfer to nitrocellulose membranes. Blots were probed with antibodies against lamins B1 and A/C (Santa Cruz Biotechnology, Inc.), PARP (BD Biosciences), ß-tubulin (Amersham Biosciences), GFP (CLONTECH Laboratories, Inc.), phospho-MLC (Thr18/Ser19), cleaved caspase 3 (Asp 175; Cell Signaling), MLC (Sigma-Aldrich), phospho-MYPT1 (Thr696; Upstate Biotechnology), MYPT1 (Covance), ROCK I (BD Biosciences), phospho-Ezrin (Thr567; Cell Signaling), Ezrin (Upstate Biotechnology), ER (Santa Cruz Biotechnology, Inc.), phospho-LIMK1(Thr508)/LIMK2(Thr505) (Cell Signaling), LIMK1 (Cell Signaling), ERK2 (provided by C.J. Marshall, The Institute for Cancer Research, London, UK), Nup153 (provided by B. Burke, University of Florida College of Medicine, Gainesville, FL), or LAP2
(provided by R. Foisner, Vienna Biocenter, Vienna, Austria) and appropriate HRP-conjugated secondary antibodies (Pierce Chemical Co.) followed by visualization with ECL (Amersham Biosciences) or SuperSignal West Femto (Pierce Chemical Co.) according to instructions and exposure to BioMax autoradiographic film (Kodak).
FACS sorting and analysis
NIH 3T3 cells were transfected with pEGFP-N3 (CLONTECH Laboratories, Inc.), pEGFP-N3 MLC-GFP, or pEGFP-N3 MLC(TASA)-GFP using Lipofectamine as described previously (Coleman et al., 2001). GFP-expressing cells were detached with trypsin, sorted, and collected using a FACSVantageSE cell sorter (BD Biosciences). The machine has a Coherent 90 C-4 argon ion laser that is tuned to 488 nm. This light was used to excite GFP, which was measured at 515545 nm.
Immunofluorescence
NIH 3T3 and primary MEFs were fixed in 4% PFA, permeabilized with 0.5% Triton X-100, and stained in PBS with goat antilamin B1 antibody (Santa Cruz Biotechnology, Inc.) at 1:500 dilution followed by FITC-conjugated donkey antigoat antibody (Jackson ImmunoResearch Laboratories) at 1:200 dilution, or with mouse antiß-tubulin (Sigma-Aldrich) at 1:200 dilution and FITC-conjugated donkey antimouse antibody (Jackson ImmunoResearch Laboratories) at 1:200 dilution. Filamentous actin structures were stained with 1:250 dilution of Texas redconjugated phalloidin (Molecular Probes). DNA fragmentation by TUNEL was done with a DeadEnd Fluorometric TUNEL kit (Promega) according to the manufacturer's instructions. Coverslips were mounted in Mowiol and visualized using a confocal microscope (model MRC1024; Bio-Rad Laboratories).
![]() |
Acknowledgments |
---|
This work was supported by American Cancer Society (RGS-04-078-01-TBE) and National Institutes of Health (CA030721) grants to M.F. Olson. Initial support provided by The Royal Society and Cancer Research UK at the Institute of Cancer Research, London, UK. The authors declare that they have no competing financial interests.
Submitted: 9 September 2004
Accepted: 17 November 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amano, T., K. Tanabe, T. Eto, S. Narumiya, and K. Mizuno. 2001. LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505. Biochem. J. 354:149159.[CrossRef][Medline]
Apel, E.D., R.M. Lewis, R.M. Grady, and J.R. Sanes. 2000. Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J. Biol. Chem. 275:3198631995.
Beaudouin, J., D. Gerlich, N. Daigle, R. Eils, and J. Ellenberg. 2002. Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell. 108:8396.[Medline]
Broers, J.L., N.M. Bronnenberg, H.J. Kuijpers, B. Schutte, C.J. Hutchison, and F.C. Ramaekers. 2002. Partial cleavage of A-type lamins concurs with their total disintegration from the nuclear lamina during apoptosis. Eur. J. Cell Biol. 81:677691.[Medline]
Bubb, M.R., I. Spector, A.D. Bershadsky, and E.D. Korn. 1995. Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments. J. Biol. Chem. 270:34633466.
Burke, B., and J. Ellenberg. 2002. Remodelling the walls of the nucleus. Nat. Rev. Mol. Cell Biol. 3:487497.[CrossRef][Medline]
Burke, B., and C.L. Stewart. 2002. Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol. 3:575585.[CrossRef][Medline]
Chiarini, A., J.F. Whitfield, U. Armato, and I. Dal Pra. 2002. Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts. J. Biol. Chem. 277:1882718839.
Clubb, B.H., and M. Locke. 1998a. 3T3 cells have nuclear invaginations containing F-actin. Tissue Cell. 30:684691.[Medline]
Clubb, B.H., and M. Locke. 1998b. Peripheral nuclear matrix actin forms perinuclear shells. J. Cell. Biochem. 70:240251.[CrossRef][Medline]
Cocca, B.A., A.M. Cline, and M.Z. Radic. 2002. Blebs and apoptotic bodies are B cell autoantigens. J. Immunol. 169:159166.
Coleman, M.L., and M.F. Olson. 2002. Rho GTPase signalling pathways in the morphological changes associated with apoptosis. Cell Death Differ. 9:493504.[CrossRef][Medline]
Coleman, M.L., E.A. Sahai, M. Yeo, M. Bosch, A. Dewar, and M.F. Olson. 2001. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat. Cell Biol. 3:339345.[CrossRef][Medline]
Croft, D.R., E. Sahai, G. Mavria, S. Li, J. Tsai, W.M.F. Lee, C.J. Marshall, and M.F. Olson. 2004. Conditional ROCK activation in vivo induces tumor cell dissemination and angiogenesis. Cancer Res. 64:89949001.
Cross, T., G. Griffiths, E. Deacon, R. Sallis, M. Gough, D. Watters, and J.M. Lord. 2000. PKC-delta is an apoptotic lamin kinase. Oncogene. 19:23312337.[CrossRef][Medline]
Feng, J., M. Ito, K. Ichikawa, N. Isaka, M. Nishikawa, D.J. Hartshorne, and T. Nakano. 1999. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J. Biol. Chem. 274:3738537390.
Gautreau, A., D. Louvard, and M. Arpin. 2000. Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J. Cell Biol. 150:193203.
Harborth, J., S.M. Elbashir, K. Bechert, T. Tuschl, and K. Weber. 2001. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114:45574565.[Medline]
Heald, R., and F. McKeon. 1990. Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell. 61:579589.[Medline]
Iwasaki, T., M. Murata-Hori, S. Ishitobi, and H. Hosoya. 2001. Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells. Cell Struct. Funct. 26:677683.[CrossRef][Medline]
Kawano, Y., Y. Fukata, N. Oshiro, M. Amano, T. Nakamura, M. Ito, F. Matsumura, M. Inagaki, and K. Kaibuchi. 1999. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J. Cell Biol. 147:10231038.
Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26:239257.[Medline]
Leung, T., X.Q. Chen, E. Manser, and L. Lim. 1996. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16:53135327.[Abstract]
McMahon, M. 2001. Steroid receptor fusion proteins for conditional activation of Raf-MEK-ERK signaling pathway. Methods Enzymol. 332:401417.[Medline]
McMullan, R., S. Lax, V.H. Robertson, D.J. Radford, S. Broad, F.M. Watt, A. Rowles, D.R. Croft, M.F. Olson, and N.A. Hotchin. 2003. Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway. Curr. Biol. 13:21852189.[CrossRef][Medline]
Mislow, J.M., J.M. Holaska, M.S. Kim, K.K. Lee, M. Segura-Totten, K.L. Wilson, and E.M. McNally. 2002a. Nesprin-1alpha self-associates and binds directly to emerin and lamin A in vitro. FEBS Lett. 525:135140.[CrossRef][Medline]
Mislow, J.M., M.S. Kim, D.B. Davis, and E.M. McNally. 2002b. Myne-1, a spectrin repeat transmembrane protein of the myocyte inner nuclear membrane, interacts with lamin A/C. J. Cell Sci. 115:6170.
Neamati, N., A. Fernandez, S. Wright, J. Kiefer, and D.J. McConkey. 1995. Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J. Immunol. 154:37883795.
Ohashi, K., K. Nagata, M. Maekawa, T. Ishizaki, S. Narumiya, and K. Mizuno. 2000. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J. Biol. Chem. 275:35773582.
Padmakumar, V.C., S. Abraham, S. Braune, A.A. Noegel, B. Tunggal, I. Karakesisoglou, and E. Korenbaum. 2004. Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton. Exp. Cell Res. 295:330339.[CrossRef][Medline]
Rao, L., D. Perez, and E. White. 1996. Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135:14411455.[Abstract]
Rosen, A., and L. Casciola-Rosen. 1999. Autoantigens as substrates for apoptotic proteases: implications for the pathogenesis of systemic autoimmune disease. Cell Death Differ. 6:612.[CrossRef][Medline]
Salina, D., K. Bodoor, D.M. Eckley, T.A. Schroer, J.B. Rattner, and B. Burke. 2002. Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell. 108:97107.[Medline]
Sampath, P., and T.D. Pollard. 1991. Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry. 30:19731980.[Medline]
Sebbagh, M., C. Renvoize, J. Hamelin, N. Riche, J. Bertoglio, and J. Breard. 2001. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell Biol. 3:346352.[CrossRef][Medline]
Shimizu, T., C.X. Cao, R.G. Shao, and Y. Pommier. 1998. Lamin B phosphorylation by protein kinase calpha and proteolysis during apoptosis in human leukemia HL60 cells. J. Biol. Chem. 273:86698674.
Shiratsuchi, A., T. Mori, Y. Takahashi, K. Sakai, and Y. Nakanishi. 2003. A presumed human nuclear autoantigen that translocates to plasma membrane blebs during apoptosis. J. Biochem. (Tokyo). 133:211218.
Spector, I., N.R. Shochet, Y. Kashman, and A. Groweiss. 1983. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science. 219:493495.[Medline]
Starr, D.A., and M. Han. 2002. Role of ANC-1 in tethering nuclei to the actin cytoskeleton. Science. 298:406409.
Starr, D.A., and M. Han. 2003. ANChors away: an actin based mechanism of nuclear positioning. J. Cell Sci. 116:211216.
Stollar, B.D., and F. Stephenson. 2002. Apoptosis and nucleosomes. Lupus. 11:787789.[CrossRef][Medline]
Sullivan, T., D. Escalante-Alcalde, H. Bhatt, M. Anver, N. Bhat, K. Nagashima, C.L. Stewart, and B. Burke. 1999. Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol. 147:913920.
Sumi, T., K. Matsumoto, and T. Nakamura. 2001. Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase. J. Biol. Chem. 276:670676.
Tran Quang, C., A. Gautreau, M. Arpin, and R. Treisman. 2000. Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes. EMBO J. 19:45654576.
Woods, D., D. Parry, H. Cherwinski, E. Bosch, E. Lees, and M. McMahon. 1997. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol. Cell. Biol. 17:55985611.[Abstract]
Zhang, Q., J.N. Skepper, F. Yang, J.D. Davies, L. Hegyi, R.G. Roberts, P.L. Weissberg, J.A. Ellis, and C.M. Shanahan. 2001. Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J. Cell Sci. 114:44854498.[Medline]
Zhang, Q., C. Ragnauth, M.J. Greener, C.M. Shanahan, and R.G. Roberts. 2002. The nesprins are giant actin-binding proteins, orthologous to Drosophila melanogaster muscle protein MSP-300. Genomics. 80:473481.[CrossRef][Medline]
Zhen, Y.Y., T. Libotte, M. Munck, A.A. Noegel, and E. Korenbaum. 2002. NUANCE, a giant protein connecting the nucleus and actin cytoskeleton. J. Cell Sci. 115:32073222.
Related Article