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Receptor-Mediated Endocytosis of Diphtheria Toxin by Cells in Culture (Clustering/Fluorescence/Video Intensification Microscopy/Clathrin-Coated Pits) JAMES H

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Receptor-Mediated Endocytosis of Diphtheria Toxin by Cells in Culture (Clustering/Fluorescence/Video Intensification Microscopy/Clathrin-Coated Pits) JAMES H Proc. NatL Acad. Sci. USA Vol. 79, pp. 2912-2916, May 1982 Cell Biology Receptor-mediated endocytosis of diphtheria toxin by cells in culture (clustering/fluorescence/video intensification microscopy/clathrin-coated pits) JAMES H. KEEN*, FREDERICK R. MAXFIELDt, M. CAROLYN HARDEGREE*, AND WILLIAM H. HABIGt *Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; tDepartment of Pharmacology, New York University Medical Center, New York, New York 10016; and Wood and Drug Administration, Bureau of Biologics, Bethesda, Maryland 20205 Communicated by Sidney Weinhouse, January 4, 1982 ABSTRACT The binding and uptake of fluorescently labeled oftoxin with cells at physiological temperature resulted in much diphtheria toxin by cells in culture has been examined byusing epi- ofthe toxin becoming inaccessible to external agents-suggesting fluorescence video intensification microscopy. Rhodamine-la- entry into the cell. Finally, extensive degradation of toxin and beled diphtheria toxin retained significant toxicity on bioassay and inhibition of protein synthesis were observed (7). in cell culture and was tested for uptake by human WI-38 and The exact mechanisms by which toxin reaches the cell cy- mouse 3T3 fibroblasts grown in culture. When added to cells at toplasm and the factors responsible for the phenomenon of re- 37rC, toxin was observed to become concentrated and internalized sistance remain unknown. We have prepared a rhodamine-la- in discrete vesicles in both cell lines. The appearance of fluores- beled derivative cent clusters could be prevented by addition of excess unlabeled of diphtheria toxin (R-DT) that retained diphtheria toxin to the medium or by addition of ATP (which has 25-50% ofthe biological activity ofcontrol preparations. Using been shown to block toxin binding to cells), indicating that the rho- this derivative and video intensification microscopy (8), we have damine-labeled toxin was binding to diphtheria toxin-specific cell found that toxin undergoes receptor-mediated endocytosis§ (9): surface binding sites. When the simultaneous uptake of rhoda- it is recognized by specific cell surface binding sites and is in- mine-labeled diphtheria toxin and fluorescein-labeled a2-mac- ternalized in endocytic vesicles. Furthermore, this process oc- roglobulin was monitored, the two proteins appeared in the same curs in both diphtheria toxin-sensitive and toxin-resistant cell clusters indicating that the toxin undergoes receptor-mediated lines. The implications of this uptake process for the toxicity of endocytosis. Despite the difference in susceptibility to diphtheria the protein are discussed. toxin ofcells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both MATERIALS AND METHODS cell lines was indistinguishable in terms of toxin required for for- Swiss mouse 3T3 clone A and human WI-38 cells were grown mation of clusters or inhibition by unlabeled toxin or by ATP. as described (10). The WI-38 cells were used between passages These results demonstrate that diphtheria toxin-specific cell sur- 16 and 34 and a single lot of calf serum (GIBCO no. 12K3101) face binding sites occur on both insensitive and sensitive cells and was used throughout these suggest that toxin is processed similarly by both cell types during studies (11). its initial cell surface binding and internalization by this pathway. DT was obtained from John Robinson ofVanderbilt Univer- The possible involvement of this uptake system in the mechanism sity. The preparation contained 20,000 minimum skin-reactive of action of diphtheria toxin in cells is discussed. doses (MRD) per Aug of protein (12) and was homogeneous on NaDodSO4 gel electrophoresis. R-DTwas prepared by reaction Extensive studies on the structure and mechanism of action of ofDT with tetramethylrhodamine isothiocyanate; DT at 10 mg/ diphtheria toxin (DT) have resulted in a detailed understanding ml in 0.05 M sodium borate (pH 9.2) was dialyzed for 24 hr at ofthe biochemical pathogenesis ofthe toxin (for review, see refs. 4°C against 100 ml of0.05 M sodium borate (pH 9.2) containing 1 and 2). DT is synthesized by Corynebacterium diphtheriae 2.0 mg of tetramethylrhodamine isothiocyanate (Cappel Lab- lysogenic for the ,3tox+ phage and is secreted as a single poly- oratories, Cochranville, PA, lot no. 8612). The labeled protein peptide chain of Mr 62,000. The toxin can be nicked by pro- was then dialyzed extensively against calcium- and magnesium- teases to yield A and B chains (Mr 21,000 and 41,000, respec- free Dulbecco's phosphate-buffered saline (Pi/NaCl) and then tively) that remain disulfide linked. The A chain catalyzes dialyzed overnight against Pi/NaCl supplemented with 1 M transfer ofthe ADP-ribose moiety ofNAD to cytoplasmic elon- NaCl. Finally, the toxin was again dialyzed against Pi/NaCl. gation factor 2 (EF-2), resulting in the arrest ofcellular protein Fluorescein-labeled a2-macroglobulin (F-a2M) was prepared synthesis. The B chain is catalytically inactive but appears to as described (13). be responsible for cell surface binding. Although EF-2 from Bioactivity of the toxins was monitored in two ways. (i) Skin eukaryotic sources is highly conserved and isolated toxin A chain reactivity was assayed by (0.1 ml) intradermal injections of di- will ADP-ribosylate EF-2 proteins from all eukaryotic sources lutions of the toxin in Pi/NaCl containing 0.2% gelatin into in cell-free extracts (3), rodents are relatively resistant to the guinea pigs (12). The area of erythema was estimated as the actions of DT (1). This resistance is continued in cell lines de- product oftwo diameters measured at right angles to each other. rived from mouse or rat tissues. (ii) In vitro protein synthesis assays were performed by using Following earlier studies of the binding of DT to sensitive confluent monolayers ofcells in 35-mm plastic dishes. The cells cells (4) and to membranes derived from both sensitive and in- were washed and dilutions of DT or R-DT were added in 1.0 sensitive cells (5), the existence ofspecific cell surface receptors on intact cells (6) has recently been documented. Incubation Abbreviations: DT, diphtheria toxin; R-DT, rhodamine-labeled diph- theria toxin; a2M, a2-macroglobulin; F-a2M, fluorescein-labeled a2M; Pi/NaCl, phosphate-buffered saline; EF-2, elongation factor. The publication costs ofthis article were defrayed in part by page charge § The phrase "receptor-mediated endocytosis" is used to describe the payment. This article must therefore be hereby marked "advertise- process by which ligands are bound to specific cell surface binding ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. sites, clustered and internalized into primary endocytic vesicles. 2912 Downloaded by guest on September 24, 2021 Cell Biology: Keen et aL Proc. Natd Acad. Sci. USA 79 (1982) 2913 ml of complete medium. After 4 hr at 37C, 0.5 /Ci of L- ['4C(U)]leucine (339 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels; N Amersham) was added and incubation was continued for 3 hr. E The cells were then washed three times with P1/NaCl (con- E taining calcium and magnesium) at 00C and solubilized by ad- dition of1.0 ml of0.1 M NaOH. The solution was acidified with 200 1.0 ml of 30% trichloroacetic acid; 75 Ag of bovine serum al- w bumin (Sigma) was added and the protein precipitate was w washed with 1.0 ml of 10% trichloroacetic acid. Pellets were solubilized with 0.5 ml of 1 M NaOH and 0.5 ml of H20; 10 ml U- of ACS (aqueous counting scintillant, Amersham) was added 0 100 and radioactivity in the samples was counted in an Intertech- nique scintillation counter. Control incubations with cyclohex- wuUR imide (5 ,ug/ml, Sigma) were routinely performed. For assay ofR-DT interaction with cultured cells, cells were plated at 1.7 x 104 cells per cm2 in 35-mm plastic dishes. The following day the dishes were washed three times with serum- -3 -4 -5 -6 -7 free medium (3TC) and R-DT was added in 1.0 ml of serum- DILUTION (log) free medium containing 0.5% bovine serum albumin (370C). After incubation for 15 or 30 min at 370C, the dishes were washed three times with serum-free medium, then washed 100 three times with Pi/NaCl, and finally were fixed by immersion U) in Pi/NaCl containing 3.7% formaldehyde. After several min- e) LLJ 80 utes, the dishes were rinsed in Pi/NaCl and mounted for _ microscopy. Observation of the cells by phase-contrast and epi-fluores- 0z C 60 cence microscopy was performed as described (8) by using a Dage MTI model 65 silicon intensifier camera. For comparison, z0 40 all images within each figure were obtained at constant manual LL -,0 settings of the camera gain, kV, and recorder video level controls. 20 Protein was assayed by the method ofLowry et aL (14) or that of Bradford (15). Bovine serum albumin or gamma globulin 0 (Bio-Rad), respectively, was used for standards. All other chem- icals were reagent grade or better. 10-4 10-3 10-2 10-1 100 101 102 TOXIN (Mg/ml) RESULTS FIG. 1. Bioactivity of control DT and R-DT. (A) Guinea pig skin Rhodamine-labeled derivatives of DT have been prepared to reactivity assay of control DT (o) and R-DT prepared with 2.0 (i) or permit visualization of toxin interaction with cells in culture. 4.0(e) mgof tetramethyirhodamine isothiocyanate (see Materials and Skin in Methods). All stock toxin concentrations were 3.0 mg/ml. (B) Effect of reactivity assays guinea pigs revealed that increasing control DT (open symbols) and R-DT (closed symbols) on protein syn- derivatization resulted in a progressive loss ofbiotoxicity (Fig. thesis in human WI-38 (o, *) or mouse 3T3 (n, *) cells.
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