Reorganization of Keratin Intermediate Filaments by the Drug-Induced Disruption of Microfilaments in Cultured Human Keratinocytes YaSl10 Kitajima, M.D., Ph.D., Shl1nichiro Ino ue, M .D. , and Hideo Yaoita, M .D. Department of Dermatology, Ji chi Mcdica l School. Tochigi-kcn, Japan It has been shown to date that a combined trea tment w ith a network of connecting star-like knots or foci . These foci nllcrotubule and microfilament inhibitors alters the cy to­ coincid ed with actin aggregates that were formed by de­ skeletal organiza tion of keratin intermediate fi laments in polymeri za tion of actin fil aments as studied by double im­ cultured HeLa, fetal mouse epidermal, and epithelial PtK2 munofluorescence using antiactin and antikeratin antibod­ cells, although neither of these inhibitors alone is able to ies. Under these conditions, no change in microtubule do so. In the present study, we fo und that di sruption of arrangement was observed. Our observations suggest that microfilaments with cytochalasin B induced a remarkable the stability and architecture of keratin fil ament organi­ reorganiza tion of keratin filaments in cultured human ke­ za tion may be supported with the microfilament rather ratinocytes, w hile disruption of microtubules with colchi­ than the microtubule cy toskeleton in cultured human ke­ cine did not affect keratin fil aments. Keratin fil ament or­ ratinocytes. J Invest D ermatol 87: 565-569, 1986 ganization in the presence of cytochalasi n B demonstrated eratin filaments arc the intermediate-sized fi l.a mcnts and microfi laments, res pectively. Recentl y, a possibility that of epithelial cells, and fo rm one of the 3 classes of acrylamide disrupts reversibl y the keratin network in PtKI cell s cytoskeletons, along with microfilaments and mi­ has been reported (1 6). Microinj ection of anti-a-keratin antibod­ crotubules, in these cell s (reviewed in [1 ,2]) . Re­ ies into epitheli al cell s has also been used for the sa m e purpose, cently, the biochemica l and immunologic character­ showing that the selective destabilization of the fil aillentous net­ KIstI CS of keratin fil am ents have been reviewed [3,4]. However, works of keratin occurs without changing ce ll shape in PtKI, the function of keratin filaments as the intermediatc fil amcnts in PtK2, and SV -40 transformed human keratinocytes (1 7). A com­ epithelial cells has not yet been clea rly defined, although thcy m ay bined treatment with microtubule and microfilam ent inhibito rs function to support mcchanically the various structures of the has also been used successfull y to induce the reorganiza ti on of cytoplasmic space [1] . the cytoskcletal array of keratin filaments in cultured HeLa and In order to characterize the dynamic functions of kcratin fila­ feta l mouse epithelial cells [1 8, 19). although disruption of micro­ ments, studies of the rearrangement of these filam ents during filaments w ith cytochalas ins [1 8,19) or microtubules with col­ mitosis have been performed [5-15). These studies have indica ted chicine derivatives [1 9,20) did not significa ntly alter the inherent that keratin intermediate fuaments are systemati ca ll y and dynam­ organi zation of keratin fi laments. However, it is of grea t interes t ically rearranged during mitosis and, although not all epithelial to note that both cytochalas in B alone and demecolcine alone cell types undergo this phenomenon, they may play some im­ altered keratin fil am ent o rga nization in epitheli al m onkey kidney portant roles in mitosis. TC7 cell s [21]. B esides these studies, some experiments have been conducted In the present study, it is shown that the disruption of micro­ to establish m ethods to disrupt the structure and fun ctions of fil aments with cytochalas in B alone induces a remarkable reor­ keratin intermediate filaments. There are no biochemical agents ganiza tion of keratin fil ament networks in cultured human ke­ that are known to specifica ll y destabilize the structure and/or ratinocytes, whil e the disruption of microtubules with colchi cine interfere with the fun ctions of keratin and other intermediate does not. filaments in a manner analogous to that of the cytostati c drugs such as colchicine and cytochalas in B, w hich act on microtubules MATERIALS AND M ETHODS Antibodies Polyclonal antibodies against keratin filam ent pro­ tein s of human epidermis, w hich were obtained by immunizing Manuscript received December 23, 1985; accep ted fo r publi ca tion April a rabbit w ith a 50 kD subunit of human epidermal keratin (1 3), 29, 1986. were used. Monoclonal antibodies (mouse IgG) against a-tubulin Supported by Grants-in-Aid for Scientific Research, nos. A-58440047 or actin were purchased from Amersham International (England). and B-59480246 from th e Ministry of Edu ca ti on, Science and Culture, As secondary antibodies, flu orescein isothiocyanate (FITC)- or Japan, and the Lydia O'Leary Memorial Foundation , Japan. Reprint requests to: Yasuo Kitajim3 , M.D., Department of Derma­ rhodamine-lissamide sulfonyl-chloride-labeled goat IgG anti­ tology, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, To­ mouse IgG, and FITC-Iabeled goat IgG antirabbit IgG, w hich ch igi-ken, Japan 329-04. were purchased fr0111 Cappel Laboratori es (Westchester, Penn­ Abbrev iations: sylvani a) , were used in the present study. DMSO: dimethylsulfoxid e FITC: fluorescei n iso thiocyanate Cell Culture After removing 111 0st of the subcutaneous tissue PBS: phos phate-buffered sa line with surgica l scissors , skin biopsies from human foreskin o r other PMSF: ph cnylm cth ancslllfonyl flu orid e sites were cut into small pieces (2 X 5 ml11) and soaked in a TPCK: L-l-tosylamide-2-phcnylcth yl chl oromcth yl ketone solution of1oo0 U / ml dispase (Godo Shusei Co., Matsudo, C hiba, 0022-202X/86/S03 .50 Copy ri ght © 1986 by The Society for Investigative Dermatology, Inc. 565 566 K ITAJIMA. INOUE. AND Y AOITA TH E JO URN A L OF IN VESTIGAT IVE DERMATOLOGY Japan) in phosphatc-buffercd sa line (PBS) for 15 min at 3re. were soaked in a solution of 0.5% Triton X-IOO, 2 mM phen­ Aftcr thi s in cubation, thc cpidermis could bc cas il y pceled off th e ylmcthancsulfonyl flu oride (PMSF) and 2 mM L-l-tosylamide-2- ' dermis with forceps. Thc epidermis was thcll in cubated in 0.25% phenylethyl chloromethyl ketone (TPCK) for 10 min at room trypsin and 0.05% EDT A for 10-15 min at 37°C to obtain di s­ temperature. perscd keratinocytcs. Approximately 105 keratinocytcs were in­ The cell s were incubated with the first antibody for 45 min at oculated on glass coversli ps in a 35-mm plastic di sh. Thc culturcs room temperature, fo ll owed by several washes in PBS. In some wcrc fcd with Dulbccco's modificd Eagle's medium supple­ specimens, double antibody labelin g was ca rried out as fo ll ows: mcnted with 20% feta l ca lf serum, O.4/kg/ml hyd rocortisonc, 10 first the ce ll s were in cubated with rabbit antibody against keratin ng/ml epidermal growth f.Ktor, 84 ng/mJ cholera toxin , 100 /kg/1ll1 for 45 min fo ll owcd by exten sive w as hing, then with the mono­ streptom ycin , and 100 U/ ml peni cillin [22 1. Thc medium was cl onal antibody against a -tubulin or actin for 45 min. After in­ changed at 2-day intervals. C ulturcs werc grown in a humidificd cubation w ith the first antibodics and w ashing, the ce ll s were atmosphere undcr 5% CO2 at 37°e. C ulturcd cell s wcrc subj ected in cubated with thc specifi c second antibodies, rinsed w ith PBS, to experiments 4-6 days after plating. air-dried, and mOllnted in glycerol. Controls for specifi city of the antibodies were routinely ca rried o ut. Photomicrographs were Treatments with Colchicine and Cytochalasin B Cytochal­ taken with Nikon flu oresccnce microscope VFD-R (Nikon, as in B and colchicin e (Sigma C hemi ca l Co., St. Louis, Missouri) werc di ssolved in OMSO and in PBS, respectivcly, and added in Tokyo, Japan). the medium to o btain the fin al conce ntrati on of 20 /kM for each. RESULTS The cells were incubated in thesc medi a for 30, 60, an d 120 min The cytoskeletal arrangement of keratin fil aments in cultured hu­ and then fixed with cold methanol (- 20°C). For control exper­ man keratinocytes, whi ch was visualized by immunofluorescence iments, the ce ll s werc in cubated in the medium containing OM SO microscopy usin g antikeratin antibodies, showed a dense mesh­ without cytochalasin B and colchicin e for the sa me periods. Cell work around the nucleus and a radiating pattern of f!lament bun­ viabili ty during experimental peri ods was ovcr 90% as judged by dl es to the ce ll periphery (F igs l a, 2a). Microtubules also showed the trypan blue exclusion tcst. an array similar to that of keratin, but they appeared to form Immunofluorescence Microscopy The cell s grown on the much thicker filam ent bundles (Fig Ib) . The flu orescence pattern coverslips were rinsed with PBS at room temperaturc, and dippcd of actin fil aments by usin g antiac tin antibody is shown Fi g 2b, for 7 min in methanol at - 20°e. T hc fi xcd cell s, on the coverslips, whi ch showed diffuse distribution of ac tin fil aments througho ut Figure 2. Double-labeled il11munofluroescence distribution of keratin ftl­ Figure 1.