Cytokeratin Coprecipitation, Involution of Intermediate Filament Networks, and Nuclear Fragmentation: a Model for Many Degenerative Diseases
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Proc. Natl. Acad. Sci. USA Vol. 91, pp. 2497-2501, March 1994 Cell Biology Truncated desmin in PtK2 cells induces desmin-vimentin- cytokeratin coprecipitation, involution of intermediate filament networks, and nuclear fragmentation: A model for many degenerative diseases K. R. Yu*, T. HIJIKATA*, Z. X. LIN*, H. L. SWEENEYt, S. W. ENGLANDERt, AND H. HOLTZER*§ Departments of *Cell and Developmental Biology, tPhysiology, and tBiochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059 Communicated by Harold Weintraub, December 10, 1993 (received for review November 1, 1993) ABSTRACT The earliest expression of truncated desmin A number of genetic and environmentally induced degen- in transfected PtK2 cells results in the formation of dispersed erative diseases of skin, liver, and nerve exhibit intracellular microprecipitates containing not only the truncated desmin, structural changes that are remarkably similar to the pathol- but also endogenous vimentin and cytokeratin proteins. ogy induced by altered desmin in PtK2 cells. We suggest that Desmin microprecipitates without vimentin or vimentin micro- these pathologies may be driven by an analogous mechanism precipitates without desmin are not observed. The micropre- in which the introduction of an altered protein acts to cipitates involving cytokeratin invariably are also positive for aggregate and precipitate related, normally interacting pro- desmin and vimentin. Over time, the precipitates enlarge into teins. 1- to 2-pm spheroids and then fuse into amorphous chimeric juxtanuclear masses that can occupy >30% of the cell volume. MATERIALS AND METHODS Concurrently, first the vimentin and then the cytokeratin networks are resorbed. The chimeric precipitates are not Full-length desmin cDNA was truncated at the unique Cvn I recognized or marked for degradation by the lysosomal system. restriction endonuclease site, within segment 2 of the highly Ultimately the cell nucleus fragments and the cell dies. Similar conserved a-helical rod domain, and subcloned as described protein complexes appear in many human and animal pathol- (16). The truncated protein contains 272-amino acid residues ogies, suggesting that a similar protein-precipitation sequence compared with 468 residues for full-length desmin. initiated by the introduction of a mutationally or environmen- PtK2 cells (American Type Culture Collection) were cul- tally altered protein molecule is at work. tured onto collagen-coated Aclar coverslips (Pro-Plastics, Linden, NJ) in 35-mm tissue culture dishes. Cells were plated Since the identification of intermediate filaments (IFs) in at an initial density of 105 cells per dish in Eagle's minimal developing myotubes >25 yr ago (1), >30 IF proteins have essential medium/10% fetal calfserum. One day afterplating, been identified. The genes for most of these have been the cells were transfected with 2 ug of DNA by standard cloned, regulatory sequences have been delineated, and the calcium phosphate precipitation methods (16, 17). conditions favoring the polymerization of these proteins into Monoclonal antivimentin antibody (clone Vim 3B4) was elongated 10-nm filaments have been studied (2-7). Still, with purchased from Boehringer Mannheim. The polyclonal an- rare exceptions (e.g., refs. 8 and 9), little is known of the tidesmin used does not react with vimentin (13). Polyclonal intracellular interactions and functions governed by these anticytokeratin, from T. Sun of New York University, does not react with other IF proteins. Polyclonal antibodies to filaments. For example, it is still unclear why newborn, nonmuscle myosin heavy chain (MHC:BioTechnology, postmitotic myoblasts up-regulate desmin synthesis before Stroughton, MA) and monoclonal antibody anti-a-actinin that ofany ofthe myofibrillar proteins (10-14) or why desmin (clone BM 75.2, Sigma) stain stress fibers. The monoclonal IFs shift from a longitudinal orientation in early myotubes to antibody antiubiquitin was obtained from Chemicon. Poly- a transverse orientation with a sarcomeric periodicity after clonal antitubulin was purchased from ICN. Rhodamine- myofibrillogenesis is advanced (10, 15). phalloidin was purchased from Molecular Probes. Affinity- To study the process of protein incorporation into IFs, purified fluorescein isothiocyanate- and rhodamine- Schultheiss et al. (16) transfected replicating myogenic cells conjugated goat anti-rabbit IgG and anti-mouse IgG and IgM with a truncated desmin cDNA. They reported that the were purchased from Jackson ImmunoResearch. truncated desmin was not incorporated into the cell's preex- The problem of bleed-through in the fluorescence micro- isting chimeric desmin+/vimentin+ network. Rather, dis- scope was minimized by running duplicate series. In one persed microprecipitates formed and grew into desmin+/ series, each of the two primary antibodies was stained with vimentin+ spheroids =2 um in diameter. The growth of the either a rhodamine- or a fluorescein-conjugated secondary spheroids was correlated with the dissolution of the preex- antibody. In the second series, the same primary antibodies isting desmin+/vimentin+ IF network. were stained in the opposite fashion. Thus each antigen was To study this process further, we have similarly transfected localized by the use of fluorescein- and rhodamine- PtK2 cells, which typically assemble separate vimentin and conjugated secondary antibodies. Confocal images were col- cytokeratin networks but do not synthesize desmin. We lected, as described in ref. 16, by using Texas Red secondary describe here a sequence of events in which the altered antibodies. Nuclei were visualized with 4',6-diamidino-2- desmin progressively diverts the endogenous IF proteins into phenylindole (DAPI) (Sigma). Sections for EM were pre- massive precipitates that lead to cell death. pared as described (1). The publication costs of this article were defrayed in part by page charge Abbreviations: IF, intermediate filament; DAPI, 4',6-diamidino-2- payment. This article must therefore be hereby marked "advertisement" phenylindole. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 2497 Downloaded by guest on September 29, 2021 2498 Cell Biology: Yu et al. Proc. Natl. Acad. Sci. USA 91 (1994) RESULTS Concurrent with the growth of spheroids, a dramatic overall thinning and retraction of the vimentin network toward the Well-spread PtK2 cells assemble robust, widespread vimen- cell center occurs. The cytokeratin network is less drastically tin and cytokeratin networks (Fig. 1 a and b). Neither reduced (compare Fig. 1 e with f). Changes in both IF network stains with antidesmin. After transfection with trun- networks are selective and not due to some generalized cated desmin cDNA, between 5 and 40%o of the cells in each cytotoxicity; double-staining with antibodies to actin, non- dish elaborate a modified desmin protein that binds antibod- muscle myosin heavy chains, a-actinin, vinculin, and tubulin ies to desmin. The expressed desmin produces a character- document normal morphological integrity ofthe stress fibers istic sequence of events, as follows. and the microtubules (data not shown). Within 12 hr, the transfected cells display large numbers of Within 48 hr after transfection, many ofthe spheroids fuse variably sized, dot-like structures (Fig. 1 c and d). All the into massive, irregularly shaped complexes (Fig. 2 a and c). dots are chimeric, binding both antidesmin and antivimentin. Confocal reconstructions ofthese bodies give the impression Neither desmin-/vimentin+ nor desmin+/vimentin- dots of links in a coiled chain (data not shown). These are often have been observed. The dots emerge at random throughout juxtanuclear in location but not always. The massive links the cytoplasm; their appearance near the nucleus does not can occupy >30%o of the cell volume and can measure up to precede their appearance at the cell periphery. At this early 15 um in length and 6 ,um in thickness. The convoluted links time after transfection, =25% of the transfected cells suffer costain precisely with antibodies to desmin and vimentin. a modest reduction in extent of the vimentin network, Anticytokeratin generally stains the more superficial regions whereas the cytokeratin network is unimpaired (see below). (Fig. 2c). Whether this reflects inaccessible cytokeratin At 24 hr after transfection, the inclusions have increased in epitopes or involves other factors remains to be seen. number and size. A gradient of chimeric inclusions appears; Additional major alterations appear in cells with prominent 1- to 2-pum spheroidal bodies immediately around the nucleus chimeric, juxtanuclear links. Over 95% (n = 200) either lose trail off to smaller dots at the cell periphery. The PtK2 their vimentin network totally or retain only small numbers spheroids physically resemble the desmin+/vimentin+ bod- of short, scattered filaments (Figs. le and 2a), while the ies seen in myotubes infected with the same cDNA (16) but cytokeratin network exhibits significant, although less ex- differ in that 60-70% also contain cytokeratin (Fig. 1 e andf). tensive, reduction (Figs. 1f and 2c). In the 48- to 96-hr FIG. 1. (a and b) Fluorescence micrographs of three PtK2 cells double-stained with antibodies to vimentin (a; rhodamine channel) and cytokeratin (b; fluorescein channel). Both IF networks extend well into the cell periphery. Asterisks mark same cells. (c and d) Micrographs of four double-stained PtK2