Prinfedin Sweden Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form resrrucd

Experimental Cell Research91 (1975) 47-56

BINDING

AND

SUBCELLULAR

LOCALIZATION

CYTOCHALASIN JANET TANNENBAUM, Departments of Microbiology

S. W. TANENBAUM,l A. F. MIRANDA

OF TRITIATED

D L. W. LO, G. C. GODMAN,

and

and Pathology, Columbia University, New York, N.Y. 10032, USA

SUMMARY Tritiated cytochalasin D (3H-CD) is rapidly taken up by monolayers of HEp-2 HeLa and rhahdomyosarcoma cells, reaching a maximum incorporation within 5 min at 37°C. Upon rinsing and refeeding, 80 % of the bound drug rapidly dissociates from the cells; the remaining 20 % is lost ‘more slowly. Binding is dose-dependent in a non-linear fashion; Scatchard plots are biphasic, suggesting binding of higher and lower affinity. Inhibitors of energy metabolism do not diminish binding of 3H-CD. The plasma membrane fraction of HEp-2 exhibits the highest specific binding activity (146 dpm/pg protein) and contains both high and low affinity binding sites. Endomembranes (microsomes) have moderate specific binding activity (35 dpm/pg protein) and appear to contain only low affinity binding sites. Nuclear, mitochondrial, and cytosol fractions exhibit low, probably negligible, binding. These results are consistent with the evidence afforded by radioautography. Selective enzyme digestions of whole cells and the plasma membrane fraction indicate that binding of CD requires proteins not exposed on the outer surface of the cell. Because electron micrographs of the plasma membrane fraction demonstrate microfilaments attached to the membrane, the binding data may be interpreted as evidence for an interaction of CD either with the subplasmalemmal microfilaments or directly with the plasma membrane.

The cytochalasins, a group of congeneric secondary fungal metabolites [1], affect a seemingly diverse number of important cellular functions such as locomotion, membrane mobility, cytokinesis, exo- and endocytosis, and transport of sugars (reviews [2-51). The unifying hypothesis that many of these phenomena depend upon the integrity of the contractile apparatus of the cell has been proposed [3], and it has been suggestedthat the cytochalasins may act directly on the cortical microfilaments [3, 6, inter alia]. However, others have inferred that the plasma mem-

1 Present address: School of Biology, Chemistry, and Ecology, SUNY, College of Environmental Science and Forestry, Syracuse, N.Y. 13210, USA. 4-751805

brane is the primary site of action of the cytochalasins [i.a. 7-111. CB, at rather high concentrations (lO-3 M), has been said to react with actin [12, 131;more recently, it has been reported [14] that CD at lO-6 M can bind to myosin. Fundamental to any understanding of the mechanism of action of these agents is information on where in the cell, and to what, cytochalasin is bound. This communication details the results of studies, hitherto reported in preliminary form [15], of the binding of tritiated cytochalasin D (3HCD) to cells of some established lines and to subcellular fractions. The sites and characteristics of CD uptake are described, together with observations on the protein nature of the putative binding components. Exptl Cell Res 91 (1975)

48

Tannenbaum et al.

MATERIALS

AND METHODS

Cell culture The cell lines utilized in this work were grown in monolayers as detailed in [4]. Human embryonal rhabdomyosarcoma cells (RD) were obtained from the American Type Culture Collection (CCL136).

Cytochalasins Cytochalasin D (CD) was isolated from one or more producing strains of Zygosporium mason2 [16]. Tritiated cytochalasin D (3H-CD) was prepared by the microwave discharge procedure of Hembree et al. [17]. In contrast to trial experiments utilizing the Wilzbach technique, the exchanged-out reaction mixtures following the cited mode of preparation exhibited little destructive heterogeneity, and consisted almost solely of 3H-CD. The labeled drug was purified by repeated preparative thin layer chromatography (in these solvents: ethyl acetate, toluene/methanol, 100/12 or 75/10, v/v) to constant specific activity (0.70 mCi/ md. (Soecific activitv was measured bv scintillation count& of 3H-CD solutions prepared-with weighed samoles of $H-CD.) As iudaed tic radio- bv- analvtical chemical assay, the 3HrCD was found to be at least 98 % homogeneous. Degradation exoeriments indicated widespread scrambling of radioactivity, predominantly in the benzyl side chain, but also in the macrolide ring moiety. Details of this preparation will be published elsewhere 1411.Stock solutions of CD in DMSO were diluted in medium or buffer solution to the requisite concentrations before use. In a test for its biological activity, carrier-free 3H-CD exhibited a titer identical to that obtained with unlabeled CD by the standard zeiosis assav (vide infra). Using the zeiosis assay, we found that 11:dihydro CD was about 12 % as potent as CD; Minato et al. [18] obtained a similar value with other bioassay methods. Competition experiments between 3H-CD and CD for binding to HeLa cell monolayers (vide infra) demonstrated ihat four times as much tritium label was bound to cells incubated with 1 ,u&ml 3H-CD as to cells incubated -. with 0.25 pug/ml 3H-CD plus 0.75 pup/ml CD. Therefore, 3H-CD is able to compete equally well (on a mole-formole basis) with unlabeled CD for binding to cells and thus represents a valid probe for CD binding sites.

Uptake of aH-CD by intact cells (1) Monolayers. Monolayers of cells olanted at 2.5 x ids cells/2~ ml/vial were grown overnight in glass scintillation vials. The growth medium was then replaced with one containing 3H-CD and replicate monolayers were further incubated for the time periods indicated in the legends. Cell-bound 3H-CD was measured as the label which remained associated with the cells following removal of the 3H-CD and two rapid rinses with chilled EBSS (Earle’s Balanced Salt Solution). Background counts were determined by carrying out these procedures on sham-planted vials not containing cells. (2) Centrifrrgatio~ assay. Cells of various origin (2 x 10e) were harvested by trypsinization and treated with 3H-CD (0.52 pgjml) hr SMT (0.25 M sucrose, 1 mM MgCl,, 10 mM Tris-HCI, pH 7.0). After 15 min at 37°C the cells were centrifuged, rinsed in chilled SMT, taken up in distilled water and sampled for protein [19] and bound label. Appropriate controls lacking cells provided background counts. Samples of untreated cells were taken for hemocytometer counts and protein determination. Uptake of 3H-CD per lo6 cells was calculated from the dom cell-bound 3HCD/pg protein and the protein vaiues per lo6 cells. Radioactivity was determined by scintillation counting of samples~dissolved in Soluene-lOO(Packard) and mixed with a toluene-Omnifluor cocktail (4 g/l, New England Nuclear). Efficiency of counting was determined by internal standardization. (3) Red blood cell zhosts. Blood cell ahosts (Tvoe 0) were prepared by lysis in 20 mOsm pho&hatk buffer: DH 7.4 f201: binding of 3H-CD was measured as described for particulate HEp-2 fractions (vide infra).

Inhibitors Solutions of the following inhibitors were prepared, in the concentrations given, in glucose-free growth medium supplemented with 10% dialvsed newborn calf serum:-?,4 dinitrophenol, 5 mM (Sigma); potassium cyanide, 2 mM; iodoacetamide, 1 mM (Siama): 2-deoxyglucose, 10 mM (Pierce Chemical). HeL> ce\i monolayers grown in glass scintillation viaIs were pretreated with inhibitor solutions for 5 min at 37°C and then exposed to 0.2 fig/ml 3H-CD in the continued presence of the inhibitors for 10 min. Cell-bound 3HCD was determined as described above.

Zeiosis assay

Radioautography

CD induces zeiosis, i.e., the protrusion of endoplasmic knobs (fig. l), in HeLa and other sensitive cells [4]. This morphological effect was used in a titration assay for CD activity. HeLa cell monolayers were exposed to a graded series of 1 :2 dilutions of CD or DMSO for 1 h at 37°C then fixed and stained with azureeosin. Azurophilic zeiotic knobs were easily recognized in the light microscope (fig. 1). The highest dilution of CD which produced detectable zeiosis in this procedure was 0.03 ,ug/ml (60 nM).

Subconfluent monolayers (3 x lo6 cells/35 mm dish) of HeLa, HEp-2, and MDBK were treated with 0.5 or 1 pg/ml of 3H-CD for 10-30 min. The cultures were then rinsed quickly in 3 changes of EBSS at 4°C blotted, quenched in propane, cooled with liquid nitrogen, then transferred to liquid nitrogen, freezedried in vacua and fixed in formaldehyde vapor. Gelated films of Ilford KS emulsion were applied to the coverslip and exposed for 2 weeks at 4°C and then processed thereafter as usual.

Exptl

Cell Res 91 (1975)

Binding of tritiated cytochalasin D

49

Fig. 1. Zeiosis in HeLa cells. Monolayers were exposed to 0.1% DMSO (left) or 0.5 ,ug/ml CD (right) in growth medium for 1 h, then fixed and stained with azure-eosin. Note in CDazurophilic knobs (arrow) treated cells. x 460.

Cell fractionation HEp-2 cells were harvested by trypsinization, suspended in growth medium, and generally washed twice with EBSS before Dounce homogenization. In nreliminary fractionation studies, the postgranular supernatant was collected after sedimentation of nuclei (225 g/10 min) and combined microsomes (14 700 g/30 r&n). The plasma membrane fraction was isolated by the method of Atkinson & Summers [21], but without use of sodium iodoacetate or sodium azide. A microsomal fraction which appeared as a fluffy band at the Tris-30% sucrose interface of the discontinuous sucrose gradient was also collected (method 1). The crude nuclear pellet obtained in this procedure was suspended in 0.88 M sucrose, 1 mM MgCI, and further purified by the procedure of Maggio et al. [22], using 1 mM MgCl, instead of CaCl, in the sucrose gradients. Mitochondria were isolated by the technique of Nass 1231. A microsomal fraction was also obtained by centrifugation of postmitochondrial supernatant [24] at 100000 g (Beckman Spinco L-2, SW 40 rotor) for 2 h (method 2). Purity of fractions was monitored by electron microscopy (Procedures in [4]).

Binding assaysfor fractions Samples of particulate fractions (except for microsomes prepared by method 2) were suspended in SMT and assaved for 3H-CD binding bv the centrifugation assay used for whole cells (vide supra). The microsomal pellet (method 2) was exposed to 3H-CD in SMT without resuspension of the pellet into the solution. A nellet of whole HEp-2 cells (sedimented at 1 000 g) was similarly treated in order to estimate the efficiency of this assay procedure compared with the standard one. Binding activity of this microsomal fraction was corrected for the reduced efficiency of binding in this nrocedure. After incubation of postgram&r super&ant with 3H-CD, bound 3H-CD was determined by gel filtration on columns of Sephadex G-25 (Pharmacia) or Biogel A-O.5 m (Bio-Rad) eluted

with 10 mM Tris-HCl, 1 mM MgCl,, pH 7.0. Macromolecular-bound 3H-CD was eluted in the void volume coincident with the protein oeak. A second ueak of unbound 3H-CD was-retarded on the column at the elution volume of control 3H-CD solutions.

Enzymatic digestion HEp-2 cells were harvested by trypsinization (0.125 % trypsin in EBSS with 0.15 % versene for 5 min at 37”C), suspended in growth medium, and washed twice in EBSS. For experiments testing the effect of trypsin on uptake of 3H-CD, cells were detached from the monolaver bv 0.15 % versene in EBSS. without use of trypsin.+Enzyme digestions were performed on intact cells suspended in HEPES-buffered EBSS for 15 min at 37°C. Cells were treated with neuraminidase in bicarbonate-buffered EBSS for 30 min at 37°C in an atmosphere of 5 % COz, 95 % air. Cells were then washed and uotake of 3H-CD (0.52 tie/ml) was measured in serum-free growth ‘medium by the centrifugation assay already described. The plasma membrane fraction was treated in a similar manner, except that digestion was carried out in 10 mM TrisHCI, 1 mM MgCl,, pH 7.4 at 37” for 15 min (neuraminidase was incubated with membrane as described for whole cells). Binding was measured in SMT (0.52 /Ag SH-CD/ml). The following enzymes were used: trvosin (Worthinaton Biochemical Corn): RNase (p&ease~free RNaseA; Sigma); pronaie I (Calbiothem): nhosnholiaase C (Cl. rxrfrinens;Worthington BiochemicalCorp.); neuraminidaseV(from Dipfoco&us pneumoniae [25]). Proteolytic activity of phospholipase C was destroyed by heating the enzyme solution to 60°C for 10 min and centrifuging to remove precipitate WI.

RESULTS Uptake studies Uptake of 3H-CD by monolayers of HeLa and HEp-2 cells was rapid and reached a maxiExptl

Cell Res 91 (1975)

50

Tannenbaum et al. had no significant effect on binding of 3HCD by HeLa cells. Uptake of 3H-CD was dose-dependent in a non-linear fashion for all of the cell lines examined (HEp-2, HeLa, RD, L, Vero, MDBK, PR-105 fibroblasts, and WIL-2). As shown forHEp-2 and RD, the curves exhibited a sharp declination in slope at about 0.2 ,ug/ ml (fig. 3 a). Scatchard plots of these data (fig. 4) were biphasic, indicating the presence of

Fig.

2. Abscissa:

1OF3/monolayer.

time (min); ordinate:

3H-CD dpm x

monolayers grown in glass scintillation vials were a treated with 0.26 ,ug/ml 3H-CD in growth medium. Cell-bound 3H-CD/monolayer was determined after , aspiration of 3H-CD medium and two rapid rinses with chilled EBSS. Points are average values for 2-3 samples. Background counts were determined on vials o o sham-planted with medium lacking cells. (a) HeLa. At 30 min (arrow), 3H-CD was removed,cellswere rinsed twice with warm (37°C) EBSS, and refed with drug-free medium. Cell-bound 3H-CD was deter250.. mined as described above. (The lower point under thelarrow indicates cell-bound 3H-CD immediately 200. after warm rinses.) (b) HEp-2. At 15 min (first arrow) 3H-CD was removed, cells were rinsed twice with warm serum-free growth medium and refed with growth medium. At 75 min (second arrow), one group of vials was rinsed and refed a second time (----); -, values without second refeeding.

0.26

0.52

I.04

2.08

. b

mum in lessthan 15 min at 37°C (fig. 2) over a range of biologically active concentrations 0 026 0.52 0.73 I .04 of CD (0.13 to 1.04 ,ug/ml). Shorter incuba- Fig. 3. Abscissa: pg/ml 3H-CD; ordinate: (a) 3H-CD tion times showed that maximum uptake was dpm x 10-3/105 cells; (6) 3H-CD dpm/,ug protein. Effect of 3H-CD concentration on binding to whole achieved within 5 min. The association of cells and membrane fractions. (a) Binding of 3H-CD 3H-CD with cells was also rapidly reversible to RD (0) and HEp-2 (0). Replicate monolayers treated in triplicate with growth medium conat 37°C when monolayers were washed and were taining the indicated concentrations of 3H-CD for refed with drug-free medium (fig. 2). Apparent 15 min at 37°C. Cell-bound 3H-CD was determined in fig. 2. Several replicate monolayers were trypresidual binding at 40 min after refeeding was as sinized for cell counts in a hemocytometer. Range also reversible if cells were refed a second bars: S.D. (b) Binding of 3H-CD to HEp-2 plasma time (fig. 2b). Uptake was apparently not membrane (0) and microsomes (0). Samples suspended in SMT containing the indicated concentraenergy dependent, because inhibitors of tions of 3H-CD were incubated 15 min at 37”C, collected by centrifugation, and rinsed with chilled energy metabolism (dinitrophenol, deoxygluSMT before determination of 3H-CD bound/pg case, iodoacetamide and potassium cyanide) protein Exptl

Cell Res 91 (1975)

Binding

of tritiated

cytochalasin D

51

more than one class of binding affinities [27]. The higher affinity binding had an apparent association constant of about 10’ l/mole for both HEp-2 and RD cells. The approximate number of high affinity 3H-CD binding sites per cell was 10’ for HEp-2 and 4 x 10’ for RD cells. Localization of binding Radioautography. With

procedures which minimized loss or diffusion of cell-bound 3HCD (see Materials and Methods), radioautograms of MDBK, HeLa, or HEp-2 cells showed grains distributed over all of the cytoplasm but with very few over the nucleus (fig. 5). If the drug were bound exclusively to the outside of the cell, there would have been a more uniform distribution of grains over the entire cell surface. Our results therefore suggested that binding might not be restricted to the outer surface of the cell, but that some 3H-CD could penetrate into the cytoplasm (An alternative interpretation of the paucity of grains over the nucleus might be that the nucleus absorbs p-rays originating in the adherent surface of the cell subjacent to the nucleus which elsewhere could reach the emulsion. However, the generalized, non-

0

I

2

3

4

5

6

7

r ( x 10-l’); ordinate: r/c (X lo-lo). Scatchard plots: binding of $H-CD to RD (0) and HEp-2 (0). Data from fig. 3a. r, moles CD/cell; c, moles free CD/l. Fig. 4. Abscissa:

Fig. 5. (a) to 3H-CD distributed developed (b) MDBK

Radioautograms of MDBK cells exposed (0.5 pg/ml) for 30 min. Silver grains are over the cytoplasm; only few grains have on the area overlying the nucleus ( x 400). cells at higher magnification treated as in

(a) ( x 900).

preferential distribution of grains over the entire cytoplasm, including the perinuclear ring, suggests that /?-emanations from the plasma membrane of the basal (adherent) aspect of the cell may not have contributed importantly to the observed pattern of distribution of grains overlying the cells. A gradient of grain density from periphery to center, such as would be expected if any considerable part of the radioactivity reaching the emulsion originated in the undersurface of the cell, is not seen. Calculations of the influence of cell thickness (depth) and anhydrous mass (density) on the self-absorption of bExptl

Cell Res 91 (1975)

52

Tannenbaum et al.

Table

1. Binding

of 3H-CD

to HEp-2

cell

fractions Fraction Whole cells Nuclear Plasma membrane Mitochondrial Microsomal Supernatant

Spec. binding act. (dpm/pg protein) 56 4 146 3: -6

Fractions were suspended in SMT containing 0.52 @g/ml 3H-CD and incubated 15 min at 37°C. Particulate fractions were collected by centrifugation at 4°C and rinsed with chilled SMT. Bound 3H-CD/pg protein was determined for each pellet. 3H-CD bound to supernatant was determined by gel exclusion chromatography (see Materials and Methods).

rays from 3H, as given by Perry [42] make it probable that only minor amounts of p-radiation can reach the emulsion from the adherent surfaces of these cells through the overlying central cytoplasm, whose profile is probably planoconvex and whose thickness is estimated to be not less than 3 ,um, and whose density is greater than that of nucleoplasm (excluding nucleolus). The possibility was therefore entertained that some radioactivity may originate in endoplasm, as well as in plasma membrane.) The distribution of grains was the same after shorter (10 min) or longer (60 min) exposure to the drug, demonstrating the absence of a time-dependent gross translocation of CD within the cell. Binding by cell fractions

Eighty to ninety percent of the CD binding activity was recovered in particulate fractions (components that sedimented at 14 700 g/30 min). Binding data for HEp-2 fractions are given in table 1. The plasma membrane, the fraction with greatest binding activity, always showed attached microfilaments (fig. 6). The dose-dependence curve for 3H-CD binding by plasma membrane (fig. 3 b) exhibited a nonExptl

Cell Res 91 (1975)

6. HEp-2 plasma membrane. Note microfilaments apparently attached to the membrane. x 48 700.

Fig.

linear pattern of higher and lower affinity binding similar to that seen in whole HEp-2 cells (fig. 3 a). In contrast, the curve for microsomes (prepared by method 1) was approximately linear without detectable high affinity binding (fig. 3b). The dissimilarity of the two curves demonstrated that binding of 3H-CD by microsomes was not due to contamination of this fraction by vesicles of plasma membrane. Table 2. Effect of enzyme digestion on binding of 3H-CD Intact cells

Plasma membrane

Percent Binding of activity control (dpm/pg binding protein)

Percent of control binding

Binding activity (dpm/pg protein)

;s 66

100 16 13 79

107 24 31 87

61 n.d.

75 n.d.

12

Untreated 100 Trypsin 96 Pronase n.d. Neuraminidase 98 Phospholipase C 75 RNase n.d.

55

Replicate samples of cells or of HEp-2 plasma membrane were pretreated with enzymes in the following concentrations: trypsin, 1.5 mg/ml; pronase, 0.5 mg/ ml; neuraminidase, 10 U/ml; phospholipase C, 1 mg/ ml; RNase, 0.5 mg/ml. After 15 min at 37°C (30 min for neuraminidase), samples were washed and assayed for binding of $H-CD (see Materials and Methods). Untreated controls were incubated similarly, but without enzymes.

Binding of tritiated cytochalasin D

-0

0.25

0.50

0.75

1.00

'2.00

Fig. 7. Abscissa: pg/ml 3H-CD; ordinate: (left) % biological response; (right) 3H-CD dpm x l0-3/monolayer. Effect of CD concentration on biological resDonses and binding of CD. Uptake of SH-?D by-HeLa monolayers (A ) was determined as described in fig. 2. Biological responses (data redrawn from ref. [4]): % cells exhibiting zeiosis (0); % cells with nuclear protrusion (0); average % loss of cell area (contraction) (A).

Nature of CD binding sites

Pre-treatment of intact HEp-2 cells with proteolytic enzymes, neuraminidase, or phospholipase C did not decrease the specific binding activity (table 2). On similar treatment of the isolated HEp-2 plasma membrane fraction, proteases greatly decreased binding activity. None of these enzyme digestions caused fragmentation or gross degradation of the plasma membrane fraction. DISCUSSION Uptake studies

The rapid uptake of 3H-CD by cells and its quick dissociation correlate well with the almost instantaneous biological effects of cytochalasins and the speedy recovery from them. Binding of aH-CD to HeLa and HEp-2 (fig. 2), and to platelets [14] is already maximal at 5 min. The rapid and complete dissociation of cell-bound 3H-CD upon refeeding of cells with drug-free medium (fig. 2b) suggests that the drug is not covalently bound to cells; prior experiments had shown that 3H-CD was

53

neither metabolized nor covalently bound to platelets [14]. Although the visible responses of the cell to CD are energy-dependent [4, 281, uptake of 3H-CD is not affected by inhibitors of energy metabolism and coupling, indicating that the binding process itself can be divorced from the energy requiring biological effects. The biphasic binding curves for 3H-CD uptake (figs 3a, 4) indicate that there are both high and low affinity sites for the drug and yield an association constant of lo7 l/ mole for the high affinity site. Comparable uptake curves have been reported for 3H-CB in several cell types, including HeLa and red blood corpuscles, from which K,, values of 1O-7 M (high affinity) and 1O-5 M (low affinity) were calculated [29]. Evidence for a high affinity site for CB in red blood corpuscles has also been adduced from equilibrium binding studies [30]. The shape of the uptake curves is reminiscent of those presented for steroid binding to cytoplasmic receptors, in which only the high affinity, saturable part was considered to represent specific binding (e.g., [31]). However, because apparent saturation of high affinity CD binding sites occurs at a lower concentration of CD than that required for maximal levels of some cellular responses (contraction and nuclear protrusion, fig. 7), low affinity binding of CD may be necessary for some of the biological effects. Lin et al. [29] similarly indicate that the low affinity sites for CB may be important for its biological action. Localization

Measurement of 3H-CD binding by subcellular fractions shows highest binding activity in plasma membrane, lower but significant binding by microsomes, and negligible binding by the remaining particulate fractions (table 1). We assume that some of the apparently internalized drug (as suggested by radioExptl Cell Res 91 (1975)

54

Tannenbaum et al.

binding activity membrane lipid content

Table

3. 3H-CD

dpm

3H-CD/pg protein

Human red blood cell ghosts 64 HEp-2 plasma membrane 146

pug protein/ /Ig lipid

and total dpm

3H-CD/pg lipid

1.1

70

1.5

219

Samples were incubated in SMT containing 0.52 pg/ ml 3H-CD for 15 min at 37”C, collected by centrifugation and rinsed with chilled SMT. 3H-CD bound/pg protein was determined; 3H-CD bound/,ug lipid was calculated from protein-lipid ratios given in [40].

autograms, fig. 5) is bound to the endomembranes, while the remainder of the 3HCD visualized in the radioautograms corresponds to drug located on (or in) the plasma membrane. Microsomal membranes appear to lack the high affinity binding sites detectable in plasma membrane (fig. 3 b) and intact cells (fig. 3a). The low binding activity measureable in the postgranular supernatant (table l), which may represent a small amount of membrane-derived component, is an apparent contradiction to the results of Lin & Spudich [32]. After exposing platelets to 3HCB and then disrupting them in 0.6 M KCI, Lin & Spudich [32] found most of the bound 3H-CB in the postgranular supernatant. However, this concentration of KC1 may well have solubilized the binding components from particulate elements, because similar ionic conditions yield myosin bound to 3HCD in the supernatant of an homogenate of whole platelets previously incubated with 3HCD [14].

Nature of binding sites The insensitivity of 3H-CD binding by whole cells to pronase and trypsin treatments, in contrast to the marked diminution of uptake by isolated plasma membrane exposed to Exptl

Cell Res 91 (1975)

proteolytic enzymes (table 2), demonstrates: (1) that somemembrane-associatedprotein(s) are required for binding of CD; and (2) that in the intact cell, the protein component(s) are located either-at the inner surface of the membrane or so embedded in themembrane as to be protected from proteolysis restricted to the outer cell:surface. These proteins may be the CD-receptor or components required for integrity of the binding site. The minimal effects of neuraminidase and phospholipase C indicate that integrity of sialic acid containing residues and phospholipids is not essential for binding of 3HCD to the plasma membrane. Enhancement of binding to cells after pronase digestion may result from an increased accessibility of binding sites.

Specificity of binding sites The high binding activity of the plasma membrane fraction accords with the lipophilic nature of the cytochalasins. Indeed, this property of CB, as well as the linear proportionality of 3H-CB uptake with drug:concentration in Chinese hamster ovary cells (in the range 0.2 to 40 ,ug/ml) was interpreted by Hauschka [33] as evidence for a partitioning of CB into the cell lipids rather than a specific binding of CB to cellular ‘receptor’ molecules. However, fig. 3a and the data of Lin et al. [29] show that neither 3H-CD nor 3H-CB binding is linear with concentration for a variety of cell types when a range of dosages below 0.2 pug/ml is included. The Scatchard plots suggest the existence, at least, of a saturable high affinity binding site (i.e. ‘receptor’) for cytochalasins (fig. 4 and [29]). Moreover, binding of 3H-CD is not proportional to lipid content (table 3) and thus is not accounted for merely by nonspecific liposolubility. Tests of the effect of delipidation on membranes should provide information on the possible role of lipids, if any, in

Binding of tritiated cytochalasin the

binding

of

agents can interact vinblastine colchicine

CD.

Certainly

strongly

lipophilic

with proteins

as

does with tubulin [34] and as may do with a protein component

of delipidated

membrane

[35].

triggers initial

a generalized and primary

contractile

D

55

event as its

effect [4]. Demonstration

of the sites and mode of binding

of 3H-CD in

the cell is a necessary step toward elaborating the mechanism of action of these remarkable fungal products.

Locus of CD action The high binding activity found in the plasma membrane argues for those models that propose the cell membrane as the primary site of action of the cytochalasins. However, microfilaments are regularly and reproducibly a part of this plasma membrane fraction (fig. 6) as they are of such fractions from HeLa, 3T3 cells [36] and Acanthamoeba castellanii [5]. These actin-like [5] microfilaments are evidently inserted into, or very closely associated with, the plasma membrane [5, 371; there is indirect evidence that myosin-like proteins may also be associated with the plasma membrane [38, 391. Cytochalasins, then, could exert their effects by binding to a component of the contractile apparatus inserted into, or partially embedded in, the plasma membrane. The molecular identity of the binding site(s) remains to be established. Preliminary experiments indicate that dialysis of the plasma membrane fraction against 0.6 M KC1 reversibly reduces the subsequently measured binding of 3H-CD to this fraction (unpub. obs.). Taken together with the demonstration that 3H-CD binds myosin [14], and that the fact that the association constant for high affinity binding of 3H-CD to cells (lo7 l/ mole) is the same as that reported for 3H-CD binding to purified muscle myosin [14], our results are compatible with the possibility that a receptor protein for CD in or at the cell membrane is a myosin-like molecule, but do not exclude the possibility that other protein(s) are involved. Ultimately, a complete elucidation of how cytochalasin works must explain the means by which the drug

This work will be submitted by J. T. in partial fulfillment of the requirements for the Ph.D. degree in the Department of Microbiology, Faculty of Pure Science, Columbia University. This work was supported by VC 76 from the ACS and Grants CA 13835 and AI 11902-01 from the USPHS.

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Binding and subcellular localization of tritiated cytochalasin D.

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