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• W2003203299 abstract "Activation of caspases by proteolytic processing is a critical step during apoptosis in metazoans. Here we use high resolution time lapse microscopy to show a tight link between caspase activation and the morphological events delineating apoptosis in cultured SF21 cells from the moth Spodoptera frugiperda, a model insect system. The principal effector caspase,Sf-caspase-1, is proteolytically activated during SF21 apoptosis. To define the potential role of initiator caspases in vivo, we tested the effect of cell-permeable peptide inhibitors on pro-Sf-caspase-1 processing. Anti-caspase peptide analogues prevented apoptosis induced by diverse signals, including UV radiation and baculovirus infection. IETD-fmk potently inhibited the initial processing of pro-Sf-caspase-1 at the junction (TETD-G) of the large and small subunit, a cleavage that is blocked by inhibitor of apoptosis Op-IAP but not pancaspase inhibitor P35. BecauseSf-caspase-1 was inhibited poorly by IETD-CHO, our data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct apical caspase. Thus, Sf-caspase-1 activation is mediated by a novel, P35-resistant caspase. These findings support the hypothesis that apoptosis in insects, like that in mammals, involves a cascade of caspase activations. Activation of caspases by proteolytic processing is a critical step during apoptosis in metazoans. Here we use high resolution time lapse microscopy to show a tight link between caspase activation and the morphological events delineating apoptosis in cultured SF21 cells from the moth Spodoptera frugiperda, a model insect system. The principal effector caspase,Sf-caspase-1, is proteolytically activated during SF21 apoptosis. To define the potential role of initiator caspases in vivo, we tested the effect of cell-permeable peptide inhibitors on pro-Sf-caspase-1 processing. Anti-caspase peptide analogues prevented apoptosis induced by diverse signals, including UV radiation and baculovirus infection. IETD-fmk potently inhibited the initial processing of pro-Sf-caspase-1 at the junction (TETD-G) of the large and small subunit, a cleavage that is blocked by inhibitor of apoptosis Op-IAP but not pancaspase inhibitor P35. BecauseSf-caspase-1 was inhibited poorly by IETD-CHO, our data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct apical caspase. Thus, Sf-caspase-1 activation is mediated by a novel, P35-resistant caspase. These findings support the hypothesis that apoptosis in insects, like that in mammals, involves a cascade of caspase activations. 3-[(3-cholamidopropyl) dimethylammonio]-propane sulfonate occluded virus amc, 7-amino- 4-methylcoumarin Apoptosis is a dynamic process by which unwanted or diseased cells are disassembled in a rapid but systematic manner. Apoptotic cells undergo a series of dramatic and characteristic alterations in morphology that include chromatin condensation, DNA fragmentation, cytoskeletal reorganization, cell shrinkage, and membrane blebbing (1Kerr J.F. Wyllie A.H. Currie A.R. Brit. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12927) Google Scholar,2Mills J.C. Stone N.L. Pittman R.N. J. Cell Biol. 1999; 146: 703-708Crossref PubMed Scopus (214) Google Scholar). These irreversible changes in cellular architecture are initiated directly or indirectly by the proteolytic activity of the caspases, a highly conserved family of cysteinyl aspartate-specific proteases that play a major role in programmed cell death (3Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 4Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 5Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 6Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 7Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar). Not surprisingly, proper regulation of caspase activity is critical to apoptotic execution. The caspases are activated from a latent proform (procaspase) by proteolytic excision of the large and small subunits that interact to generate the active enzyme. Procaspase processing occurs through proximity-induced autoactivation or by the activity of other proteases, including caspases (3Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 4Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). In mammals, apoptotic signaling initiates a caspase cascade wherein activated initiator caspases proteolytically activate downstream effector caspases (6Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 7Cryns V. Yuan J. Genes Dev. 1998; 12: 1551-1570Crossref PubMed Scopus (1160) Google Scholar, 8Salvesen G.S. Dixit V.M. Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1943) Google Scholar). Initiator caspases possess long prodomains that interact with diverse proteins that regulate protease activation. In contrast, effector caspases have short prodomains. Initiator and effector caspases often exhibit different substrate specificities in vitro (5Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 9Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar). In invertebrates, programmed cell death plays a critical role in development, control of DNA damage, and defense of pathogens, including viruses (10Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar, 11Clem R.J. Hardwick J.M. Miller L.K. Cell Death Differ. 1996; 3: 9-16PubMed Google Scholar, 12Nordstrom J. Abrams J.M. Cell Death Differ. 2000; 7: 1035-1038Crossref PubMed Scopus (32) Google Scholar). Caspases are required for apoptosis in insects, like that in mammals. However, the mechanisms by which caspases are activated and the hierarchy of apical and effector caspases are still unclear (10Bergmann A. Agapite J. Steller H. Oncogene. 1998; 17: 3215-3223Crossref PubMed Scopus (109) Google Scholar, 13Abrams J.M. Trends Cell Biol. 1999; 9: 435-440Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 14Kumar S. Doumanis J. Cell Death Differ. 2000; 7: 1039-1044Crossref PubMed Scopus (130) Google Scholar). On the basis of sequence similarity and biochemical activity, seven caspases have been identified in Drosophila melanogaster (Order Diptera) (15Vernooy S.Y. Copeland J. Ghaboosi N. Griffin E.E. Yoo S.J. Hay B.A. J. Cell Biol. 2000; 150: 69-75Crossref PubMed Google Scholar).Drosophila DRONC and DCP-2/DREDD possess long prodomains and by analogy to mammalian caspases are candidates as initiator caspases (16Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4307-4312Crossref PubMed Scopus (240) Google Scholar, 17Chen P. Rodriguez A. Erskine R. Thach T. Abrams J.M. Dev. Biol. 1998; 201: 202-216Crossref PubMed Scopus (183) Google Scholar). DCP-1, drICE, and DECAY contain short prodomains and therefore are likely effector caspases (18Song Z.W. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (254) Google Scholar, 19Fraser A.G. Evan G.I. EMBO J. 1997; 16: 2805-2813Crossref PubMed Scopus (171) Google Scholar, 20Dorstyn L. Read S.H. Quinn L.M. Richardson H. Kumar S. J. Biol. Chem. 1999; 274: 30778-30783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Caspases have also been identified and characterized from lepidopteran insects (moths and butterflies). In particular, Sf-caspase-1 is the principal effector caspase of SF21 cells (21Ahmad M. Srinivasula S.M. Wang L.J. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), an established cell line from the nocturnal moth Spodoptera frugiperda (Order Lepidoptera). These invertebrate cells have been used extensively for studies on apoptosis because of their sensitivity to diverse death stimuli, including baculovirus infection, UV radiation, and overexpression of proapoptotic genes (i.e. Drosophila reaper, hid, and grim) and their response to known apoptotic regulators such as P35 and IAP (23Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (710) Google Scholar, 24Bertin J. Mendrysa S.M. LaCount D.J. Gaur S. Krebs J.F. Armstrong R.C. Tomaselli K.J. Friesen P.D. J. Virol. 1996; 70: 6251-6259Crossref PubMed Google Scholar, 25Manji G.A. Hozak R.R. LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 4509-4516Crossref PubMed Google Scholar, 26LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 1530-1537Crossref PubMed Google Scholar, 27Vucic D. Seshagiri S. Miller L.K. Mol. Cell. Biol. 1997; 17: 667-676Crossref PubMed Scopus (63) Google Scholar, 28Vucic D. Kaiser W.J. Harvey A.J. Miller L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10183-10188Crossref PubMed Scopus (189) Google Scholar, 29Vucic D. Kaiser W.J. Miller L.K. Mol. Cell. Biol. 1998; 18: 3300-3309Crossref PubMed Scopus (188) Google Scholar). Pro-Sf-caspase-1, which contains a short prodomain, is activated by sequential proteolytic cleavages that are initiated only upon apoptotic signaling. The first cleavage occurs between the large and small subunit at the caspase-recognition site TETD↓G, which is also conserved in Drosophila DCP-1 and drICE. This initial cleavage event is blocked by baculovirus Op-IAP but is insensitive to the pancaspase inhibitor P35 (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Op-IAP functions upstream from P35 to block apoptosis in Spodoptera (25Manji G.A. Hozak R.R. LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 4509-4516Crossref PubMed Google Scholar, 30Seshagiri S. Miller L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13606-13611Crossref PubMed Scopus (145) Google Scholar). Thus, it has been hypothesized that the first step in pro-Sf-caspase-1 activation is mediated by an apical caspase that is distinguished by its novel resistance to P35 (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). To define the mechanism by which Sf-caspase-1 is activated upon apoptotic signaling and thereby gain insight into regulation of invertebrate effector caspases, we characterized the protease activity responsible for pro-Sf-caspase-1 activation. By using time lapse video microscopy of SF21 cells, we observed an exact correlation between the morphological hallmarks of apoptosis and caspase activation. We report here that peptide-based fluoromethyl ketone inhibitors potently blocked SF21 apoptosis induced by multiple signals. In particular, zVAD-fmk and IETD-fmk prevented the initial proteolytic processing of pro-Sf-caspase-1 at TETD↓G. BecauseSf-caspase-1 itself was inhibited poorly by IETD-CHO, our data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct caspase, designated Sf-caspase-X. On the basis of these data, we concluded that the P35-insensitive activity of Sf-caspase-X is responsible for caspase activation in Spodoptera SF21 cells and that insects, like mammals, use a cascade of caspase-mediated events to execute apoptosis. S. frugiperda IPLB-SF21 (31Vaughn J.L. Goodwin R.H. Thompkins G.L. McCawley P. In Vitro. 1977; 13: 213-217Crossref PubMed Scopus (1002) Google Scholar) cells and Trichoplusia ni TN368 cells (32Hink W.F. Nature. 1970; 225: 466-467Crossref Scopus (453) Google Scholar) were propagated in TC100 growth medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories) and 2.6 mg of tryptose broth/ml. SF21 cells were transfected as described previously (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In brief, plasmid DNA in TC100 was mixed with N-[1-(2, 3-dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl sulfate liposomes for 30 min at ambient temperature. The transfection mixture was added to cell monolayers previously washed with TC100. After 4 h of gentle rocking, the transfection mixture was replaced with supplemented TC100. Transfection efficiencies ranged from 60 to 80% as judged by lacZexpression in control plates. SF21 cell monolayers were UV-B irradiated for 10 min at room temperature by using a Blak Lamp (UVP, Upland, Calif) as described previously (33Hozak R.R. Manji G.A. Friesen P.D. Mol. Cell. Biol. 2000; 20: 1877-1885Crossref PubMed Scopus (58) Google Scholar). For infection, cell monolayers were inoculated with extracellular budded virus at the indicated multiplicity of infection. Yields of infectious virus were measured by standard plaque assay using apoptosis-resistant TN368 cells. Wild-type L-1 AcMNPV (p35 +,iap −) (34Lee H.H. Miller L.K. J. Virol. 1978; 27: 754-767Crossref PubMed Google Scholar) and AcMNPV recombinants wt/lacZ (p35 +, iap −) (35Hershberger P.A. Dickson J.A. Friesen P.D. J. Virol. 1992; 66: 5525-5533Crossref PubMed Google Scholar), vΔp35 (p35 −,iap −) and vΔp35/lacZ (p35−,iap −) (36Hershberger P.A. LaCount D.J. Friesen P.D. J. Virol. 1994; 68: 3467-3477Crossref PubMed Google Scholar), and vOp-IAP (p35−,iap +) (25Manji G.A. Hozak R.R. LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 4509-4516Crossref PubMed Google Scholar) were described previously. SF21 cells were plated onto glass coverslips mounted within 35-mm culture dishes. After cell attachment, growth medium was replaced, and the cells were UV irradiated or inoculated with virus as described. After a 2-h recovery period at 27 °C, mineral oil was added to prevent evaporation. Cells were viewed on a Nikon (Tokyo, Japan) Diaphot microscope using a 100× oil emersion objective lens. Video images were obtained at the indicated intervals with a Photometrics Series 300 or Micromax digital camera. Images were background-subtracted and contrast-enhanced. QuickTime movies were produced using Adobe Premiere 5.1 using Cinepak compressor. Scale and time of compression are indicated. Levels of apoptosis induced in SF21 cell monolayers were determined by counting both apoptotic and viable, nonapoptotic cells using a Zeiss Axiovert 135TV phase contrast microscope (magnification, 200×) equipped with a digital camera and IP Lab Spectrum P software. Cells undergoing plasma membrane blebbing and/or cell body fragmentation were scored as apoptosis; both hallmarks were readily distinguished from viable cells (see Fig. 1). The mean ± standard deviation was calculated at the indicated times from the percentage of apoptotic cells of at least six evenly distributed fields of view and included from 1500 to 6000 cells. Irreversible fluoromethyl ketone peptide inhibitors z-(benzyloxycarbonyl)-DEVD-fmk,z-IETD-fmk, z-VAD-fmk, and z-FA-fmk (Calbiochem, San Diego) dissolved in Me2SO were mixed in supplemented TC100 and added to SF21 monolayers at the indicated concentrations. Cells were irradiated in the presence of peptide analogues and maintained at 27 °C. During infection, peptide analogues were added 1 h after inoculation. Me2SO vehicle was used as control. Whole cell lysates or purified proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred to membranes. To detect Sf-caspase-1, immunoblots were incubated with a 1:2,000 dilution of α-Sfcasp1 (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and goat anti-rabbit immunoglobulin G (Pierce) conjugated to alkaline phosphate. Color development was as described previously (36Hershberger P.A. LaCount D.J. Friesen P.D. J. Virol. 1994; 68: 3467-3477Crossref PubMed Google Scholar). Escherichia coli strain BL21 (DE3) cells were induced with IPTG (isopropyl-β-d-thiogalactopyranoside) for overexpression from Sf-caspase-1-encoding pET plasmids (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). C-terminal His6-tagged proteins were purified by nickel (Ni+2) affinity chromatography as described previously (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,24Bertin J. Mendrysa S.M. LaCount D.J. Gaur S. Krebs J.F. Armstrong R.C. Tomaselli K.J. Friesen P.D. J. Virol. 1996; 70: 6251-6259Crossref PubMed Google Scholar). Isolated proteins were >90% homogenous as determined by SDS-polyacrylamide gel electrophoresis and Colloidal Burst Coomassie G Stain (Z axis). Protein concentrations were determined by using the Bio-Rad Protein Assay (Bio-Rad). Sf-caspase-1 activity was measured in reactions (20 μl) containing 25 mm HEPES, pH 7.5, 1 mm EDTA, 0.1% CHAPS,1 10% sucrose, 10 mm dithiothreitol, and 10 μm tetrapeptide substrates Ac-IETD-amc or Ac-DEVD-amc (Biomol Research). Accumulation of fluorescent product (amc) was monitored by using a Molecular Dynamics Biolumin 960 Kinetic Fluorescence/Absorbance microplate reader (excitation, 360 nm; emission, 465 nm) at 30-s intervals for 30 min. Rate of product formation was obtained from the linear portion of the reaction curves within the first 10% of substrate depletion and averaged for triplicate assays. For inhibition assays, increasing concentrations of tetrapeptide aldehydes Ac-IETD-CHO or Ac-DEVD-CHO (Biomol Research) were incubated with 200 fmol of purifiedSf-caspase-1. After 1 h at ambient temperature, substrate Ac-DEVD-amc (10 μm) was added, and residual caspase activity was measured as described above. Stained gels and immunoblots were scanned at a resolution of 300 dots per inch by using a Hewlett Packard ScanJetIIcx. The resulting files were printed from Adobe Photoshop 3.0 and Illustrator 7.0 by using a Tektronics Phaser 450 dye sublimation printer. Although many cellular components involved in apoptosis have been identified, little is known about the kinetics and morphological events of cell dismemberment. Cultured cells provide a unique view into the morphology of programmed cell death. In particular, SF21 cells from the moth S. frugiperda provide a useful model system for studies on both biochemical and morphological aspects of apoptosis (21Ahmad M. Srinivasula S.M. Wang L.J. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 23Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (710) Google Scholar, 25Manji G.A. Hozak R.R. LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 4509-4516Crossref PubMed Google Scholar, 27Vucic D. Seshagiri S. Miller L.K. Mol. Cell. Biol. 1997; 17: 667-676Crossref PubMed Scopus (63) Google Scholar, 30Seshagiri S. Miller L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13606-13611Crossref PubMed Scopus (145) Google Scholar, 33Hozak R.R. Manji G.A. Friesen P.D. Mol. Cell. Biol. 2000; 20: 1877-1885Crossref PubMed Scopus (58) Google Scholar, 37Seshagiri S. Miller L.K. Curr. Biol. 1997; 7: 455-460Abstract Full Text Full Text PDF PubMed Google Scholar, 38Cartier J.L. Hershberger P.A. Friesen P.D. J. Virol. 1994; 68: 7728-7737Crossref PubMed Google Scholar, 39Clem R.J. Miller L.K. Mol. Cell. Biol. 1994; 14: 5212-5222Crossref PubMed Scopus (496) Google Scholar, 40Jones G. Jones D. Zhou L. Steller H. Chu Y. J. Biol. Chem. 2000; 275: 22157-22165Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). SF21 cells are especially attractive because of their sensitivity to diverse apoptotic stimuli and their classical apoptotic response, which includes degradation of chromosomal DNA into nucleosomal-sized fragments and vigorous membrane blebbing. Here, we used time lapse microscopy to document SF21 morphological events during apoptosis as a means to link them with intracellular biochemical processes. SF21 cells rapidly succumb to UV radiation-induced apoptosis, which consumes >90% of a culture within 9 h (25Manji G.A. Hozak R.R. LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 4509-4516Crossref PubMed Google Scholar, 33Hozak R.R. Manji G.A. Friesen P.D. Mol. Cell. Biol. 2000; 20: 1877-1885Crossref PubMed Scopus (58) Google Scholar). Because of the large size (15–20 μm) and well defined nucleus of these cells, nuclear and cytoplasmic events during apoptosis were readily discerned by time lapse video microscopy (Fig. 1; see also Video 1 in supplementary material). The first sign of UV radiation-induced alterations was chromatin condensation, which included formation of multiple opaque or dense bodies (2–3 μm in diameter) within the nucleus (Fig. 1). The appearance of these spheroidal inclusions coincided with the early activation of cellular caspases 2–3 h after UV irradiation (33Hozak R.R. Manji G.A. Friesen P.D. Mol. Cell. Biol. 2000; 20: 1877-1885Crossref PubMed Scopus (58) Google Scholar), which is consistent with caspase-mediated detachment of chromatin from the nuclear envelope and retraction into nuclear bodies (41Lazebnik Y.A. Takahashi A. Moir R.D. Goldman R.D. Poirier G.G. Kaufmann S.H. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9042-9046Crossref PubMed Scopus (483) Google Scholar, 42Rao L. Perez D. White E. J. Cell Biol. 1996; 135: 1441-1455Crossref PubMed Scopus (513) Google Scholar, 43Orth K. Chinnaiyan A.M. Garg M. Froelich C.J. Dixit V.M. J. Biol. Chem. 1996; 271: 16443-16446Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). During this early period, other transformations were observed within the nucleus, including rapid migration of small particulate bodies and formation of vesicular-like structures. Immediately thereafter, apoptotic blebbing was uniformly initiated over the cell surface, upon which abundant microvilli-like structures were still observed. Membrane blebs first appeared as small rounded protrusions but grew rapidly to produce long extensions that ultimately detached from the main cell body to form apoptotic bodies. These vesicles were translucent or opaque, suggesting that their contents varied. Blebbing lasted for 30–45 min and consumed the cell, leaving behind a dense corpse. By 9–12 h after irradiation, only free floating apoptotic bodies and cell corpses remained (see Fig.4 A, panel ii). SF21 cells are also highly sensitive to baculovirus-induced apoptosis. AcMNPV mutants that lack functional apoptotic suppressors (p35 or iap) cause widespread apoptosis that severely restricts virus yields in part because of premature host cell death (23Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (710) Google Scholar, 26LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 1530-1537Crossref PubMed Google Scholar, 35Hershberger P.A. Dickson J.A. Friesen P.D. J. Virol. 1992; 66: 5525-5533Crossref PubMed Google Scholar, 44Clem R.J. Miller L.K. J. Virol. 1993; 67: 3730-3738Crossref PubMed Google Scholar). Time lapse microscopy of SF21 cells inoculated with the p35 deletion mutant vΔp35 (Fig. 2; see also Video 2 in supplementary material) revealed that the morphological hallmarks of virus-induced apoptosis were similar to those induced by UV radiation. However, vΔp35-induced apoptosis occurred later and was less synchronous. The first signs of apoptosis included formation of dense, refractile material within and around the inside edge of hypertrophied nuclei (Fig. 2, B and C). The appearance of this intranuclear density coincided with caspase activation, which begins 9 and 12 h after infection (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 26LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 1530-1537Crossref PubMed Google Scholar). Because nuclear aggregates were absent in cells infected with wild-type virus that encodes caspase inhibitor P35 (see below), it is likely that this material is condensed chromatin resulting from caspase activity. Soon after these nuclear events, apoptotic blebbing was initiated. Blebbing initiated 9–19 h after infection (Fig. 2; see also Video 2 in supplementary material). This asynchrony may be due to variations in the timing of virus-induced apoptotic signaling. Blebbing was invariably followed by an unusual and striking series of fusions in which apoptotic bodies of an individual cell form a single spherical mass of vesicles. Although the mechanism for this resurrection-like process is unknown, it may involve virus-encoded surface proteins that mediate membrane fusion (45Monsma S.A. Blissard G.W. J. Virol. 1995; 69: 2583-2595Crossref PubMed Google Scholar). During infection, baculovirus P35 prevents premature host cell death by blocking apoptosis. The role that P35 plays in prolonging cell survival and contributing to virus productivity is dramatically illustrated by time lapse microscopy of wild-type AcMNPV-infected SF21 cells (Fig.3; see also Video 3 in supplementary material). The first visible signs of infection occurred 9–12 h after inoculation, at which time the nucleus enlarged and the cell surface became ruffled. Extracellular budded virus, the first of two morphologically and temporally distinct forms of infectious virus (46Friesen P.D. Miller L.K. Knipe D.M. Howley P.M. Field's Virology. 4th Ed. Lippincott-Raven Publishers, Philadelphia, PA2001Google Scholar), is shed in abundance at 9–20 h and probably accounts for the vesicular, nonapoptotic protrusions at the cell surface. During this period, host caspases are activated and subsequently inhibited by newly synthesized P35 (22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 26LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 1530-1537Crossref PubMed Google Scholar, 36Hershberger P.A. LaCount D.J. Friesen P.D. J. Virol. 1994; 68: 3467-3477Crossref PubMed Google Scholar). The second and largest virus particle, occluded virus (OV), appeared on the inside edge of the hypertrophied nuclei beginning 22 h after infection (Fig. 3 C). Composed of nucleocapsids embedded within a matrix of the protein polyhedrin (46Friesen P.D. Miller L.K. Knipe D.M. Howley P.M. Field's Virology. 4th Ed. Lippincott-Raven Publishers, Philadelphia, PA2001Google Scholar), these polyhedral particles expanded to occupy the entire nucleus (Fig. 3 D). The number, size (1–3 μm dia), and shape of OV particles varied between cells. OV are not produced unless apoptosis is blocked (23Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (710) Google Scholar, 35Hershberger P.A. Dickson J.A. Friesen P.D. J. Virol. 1992; 66: 5525-5533Crossref PubMed Google Scholar). Finally, in a dynamic process that resembled necrosis, OV-containing cells expanded and ruptured (Fig. 3; see also Video 3 in supplementary material). Thus, as illustrated, the virus' apparent strategy is to prolong cell survival long enough for progeny maturation, whereupon lysis facilitates virus dissemination. The morphological events delineating apoptosis in SF21 cells coincided with the early activation of caspases (21Ahmad M. Srinivasula S.M. Wang L.J. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 26LaCount D.J. Friesen P.D. J. Virol. 1997; 71: 1530-1537Crossref PubMed Google Scholar, 33Hozak R.R. Manji G.A. Friesen P.D. Mol. Cell. Biol. 2000; 20: 1877-1885Crossref PubMed Scopus (58) Google Scholar). Current evidence suggests that multiple caspases participate in SF21 apoptosis. To define the in vivo role of initiator and effector caspases in this invertebrate system, we first tested the anti-apoptotic activity of peptide inhibitors that target the caspases, including the membrane permeable peptides DEVD-fmk and IETD-fmk. Both tetrapeptides were potent inhibitors of apoptosis induced by either UV radiation or baculovirus infection (Fig. 4). Incubation of UV irradiated cells with either tetrapeptide prevented all morphological signs of apoptosis, which was widespread in untreated cultures (Fig. 4 A). Both peptides also blocked apoptosis induced by AcMNPV p35 deletion mutant vΔp35 (Fig. 4 B). At the highest extracellular dose tested (200 μm), DEVD- and IETD-fmk reduced apoptosis to less than 5%. At lower concentrations (30–50 μm), IETD-fmk was two to three times more effective than DEVD-fmk. In contrast, the control fluoromethyl ketone FA-fmk failed to affect apoptosis induced by either death stimulus (data not shown). The intracellular concentration of each peptide inhibitor was not determined. In addition to promoting cell survival, the caspase inhibitory peptides restored baculovirus multiplication. Upon treatment with DEVD- and IETD-fmk, vΔp35-infected SF21 cells accumulated OV particles at levels comparable with" @default.
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