Proc. Natl. Acad. Sci. USAVol. 83, pp. 2894-2898, May 1986Cell Biology

Regulation of cytoskeletal proteins involved in cell contactformation during differentiation of granulosa cells onextracellular matrix

(vincuflin/a-actinin/actin/steroidogenesls/interceelular communication)

AvRi BEN-ZE'EV* AND ABRAHAM AMSTERDAMtDepartments of *Genetics and tHormone Research, The Weizmann Institute of Science, Rehovot, 76100, Israel

Communicated by Elizabeth D. Hay, December 2, 1985

ABSTRACT The organzation and the expression of cyto-skeletal proteins involved in determining cell contact and shapewere analyzed in granulosa cells during their differentiation onextracellular matrix (ECM)-coated culture plates. Ratgranulosa cells from preovulatory follicles displayed an epi-thelial shape onECM and formed multilayered aggregates withnumerous gap junctions between neighboring cells. These cellshad few actin cables and often only a diffuse pattern of actinand a low amount of vinculin in very thin focal adhesion sites.In contrast, cells grown on plastic formed a monolayer of fiatcells with a reduced number of gap junctions but with numer-ous stress fibers and abundant large vinculin-containing focalcontacts. On ECM, the cells were stimulated to produce highlevels of progesterone, while only trace amounts of the steroidaccumulated in cells on plastic dishes. Two-dimensional gelelectrophoretic analysis of [35Smethionine-labeled cells re-vealed a dramatic decrease in vinculin, a-ain, and actinsynthesis in cells grown on ECM, as compared to cells grownon plastic, while the synthesis of the tubulins and of theintermediate filament protein vimentin remained unchanged.RNA blot analysis showed a marked decrease in actin mRNAlevels in cells from ECM plates, while the level of tubulinmRNA remained essentially unchanged. It is concluded that thedifferentiation of granulesa cells on ECM in vitro is associatedwith changes in cell shape and cell contacts add that suchchanges in cell morphology are accompanied by simultaneousalterations in the organization and expression of cytoskeletalproteins that are involved in determining these cellular struc-tures.

Numerous lines of evidence suggest that important growth-related cellular functions such as cell proliferation (1),expression of the differentiated phenotype (2-7), response totissue-specific hormones (8), and tissue morphogenesis dur-ing embryonic development (9, 10) are modulated throughchanges in cell shape (for reviews, see refs. 11 and 12). Sincecytoskeletal elements are responsible for determining cellmorphology and shape, our studies on cell configuration-related gene expression (13-17) were directed toward inves-tigating the possible existence of a linkage between theexpression of genes coding for cytoskeletal proteins and theorganization in the cell of the respective proteins (for re-views, see refs. 18 and 19).The extracellular matrix (ECM), a natural substrate pro-

duced by cells, affects cell proliferation, migration, anddifferentiation in vivo (20-23). The culturing of cells on ECMwas shown to enhance cell differentiation (7, 9) and also toaffect cell shape via reorganization of the cytoskeletal net-works (24, 25). In previous studies, it was found that

granulosa cells associated with the basement membrane, inthe preovulatory follicle, contained a higher density ofreceptors to luteinizing hormone and a higher steroidogenicactivity as compared to more distal cell layers (26, 27). In thisreport, we studied the differentiation of granulosa cells onECM produced by corneal endothelial cells. We show thatthe morphological changes induced in granulosa cells duringtheir differentiation on ECM is accompanied by a coordinat-ed regulation of the synthesis of cytoskeletal proteins in-volved in the formation of cell contacts and in determiningcell shape.

MATERIALS AND METHODS

Granulosa Cell Culture and Radioactive Labeling. Granu-losa cells were prepared from 26-day-old rats that wereprimed for 48 hr with pregnant mare serum gonadotropin (15international units). The ovaries were removed into McCoy's5A medium, containing 4 mM L-glutamine, 100 units ofpenicillin per ml, and 100 pkg of streptomycin sulfate per ml,and they were sequentially incubated for 15 min at 370C with6.8 mM EGTA/0.5 M sucrose to disrupt the intercellular gapjunctions (28). Granulosa cells were released by puncturingthe follicles with a 27-gauge needle. Cells were sedimented at500 x g for 20 min. About 106 viable granulosa cells wereseeded in 1 ml of McCoy's medium containing 5% fetalbovine serum, but without sucrose and EGTA, in 35-mmdiameter Falcon dishes. Dishes and coverslips coated with anextracellular matrix derived from bovine corneal endothelialcells (22) were kindly provided by I. Vlodavsky (The HebrewUniversity, Jerusalem). Cell number did not increase duringthe culturing of cells at this density on the various substrata.Cells were labeled with 100 ACi of L-[35S]methionine per ml(>1250 Ci/mmol; 1 Ci = 37 GBq; Amersham, U.K.) inmethionine-deficient Dulbecco's modified Eagle's mediumcontaining 5% dialyzed fetal bovine serum. The labelingperiod was generally 3 hr. Similar results were obtained bylabeling the cells for 1 hr or for 24 hr.

Progesterone and Cyclic AMP Assays. To assess the bio-chemical differentiation of granulosa cells, progesteroneaccumulation was determined in cultures of cells grown onECM-coated and uncoated dishes. The steroid was measured(in triplicate) by a radioimmunoassay (29). Total cyclic AMPaccumulation in cells and incubation media was determined(in triplicate) by a protein binding assay (30).

Cell Fractionation and Polyacrylamide Gel Electrophoresisof Proteins. The [35S]methionine-labeled cells were extractedwith a buffer containing 0.6 M KCl, 0.5% Triton X-100, 14mM 2-mercaptoethanol, 2.5 mM EGTA, 1 mM phenylmeth-ylsulfonyl fluoride, and 10 mM Hepes, pH 7.4 (31). The

Abbreviation: ECM, extracellular matrix.

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Triton high-salt-soluble fraction was concentrated by ethanolprecipitation (3 vol). The Triton high-salt-insoluble fractionenriched in intermediate filament proteins was dissolveddirectly in lysis buffer A (32) and equal amounts of trichloro-acetic acid-precipitable radioactive proteins were analyzedby two-dimensional isoelectrofocussing and NaDodSO4 gelelectrophoresis (32). Immunoblotting of proteins, separatedby two-dimensional gel electrophoresis and transferred tonitrocellulose paper (33), was performed with a rabbit anti-a-actinin antibody and 125I-labeled goat anti-rabbit IgG.Immunofluorescence and Electron Microscopy. Immuno-

fluorescent staining was performed with a monoclonal anti-body against chicken gizzard vinculin and with rhodamine-labeled phalloidin for the staining of actin filaments. Cellswere permeabilized for 1 min before immunostaining with abuffer containing 2.5mM EGTA, 2.5mM MgCl2, 0.5% TritonX-100, and 50 mM 2-(N-morpholino)ethanesulfonic acid, pH6.0, and were then fixed with 3.5% formaldehyde in phos-phate-buffered saline. The second antibody for vinculinstaining was rhodamine-labeled goat anti-mouse IgG. Allantibodies and purified chicken gizzard vinculin were kindlyprovided by Benjamin Geiger (The Weizmann Institute,Rehovot). For electron microscopic examination, cultureswere fixed with 3% glutaraldehyde and 1% formaldehyde in0.1 M cacodylate buffer (pH 7.4), post-fixed in 1% OS04 in thesame buffer, stained en bloc with uranyl acetate, and em-bedded in situ in Epon. Ultrathin sections were preparedfrom doubly embedded material, which was stained withuranyl acetate and lead citrate and examined in a JEOL-100Belectron microscope operated at 80 kV. The number of gapjunctions was determined in 25 representative electron mi-crographs (x 17,000) from cells cultured on plastic or ECMfor 72 hr.RNA Extraction and RNA Blot Hybridization. Poly(A)+ and

poly(A)- RNA were isolated from the cytoplasm of cells asdescribed in detail (31). Equal amounts of RNA from eachsample were separated on agarose/formaldehyde gels andwere transferred to nitrocellulose paper. The RNA blots werehybridized to 32P-labeled nick-translated DNA of plasmidscontaining actin or tubulin sequences.

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RESULTS

Morphological Characterization of Granulosa Cells Grownon ECM and on Plastic Substrata. Granulosa cells from26-day-old gonadotropin-primed rats were seeded on eitherplastic tissue culture dishes or on ECM-coated dishes. Thecells seeded on ECM attached and started to spread by 3-6hr, but it took between 16 and 24 hr for granulosa cells onplastic dishes to adhere to the substrate. After 2 days, thecells formed a monolayer of flattened cells on plastic (Fig.LA), while on ECM the cells were densely packed and oftengrew in several layers (Fig. 1B). Cells grown on uncoateddishes displayed a flattened profile in ultrathin sections.Occasionally, stress fibers were observed in the cytoplasm ofthese cells extending toward the surface of the plastic dish(Fig. 1C). In contrast, cells grown on ECM appeared morespherical and often sent extensions from one cell to another.In these extensions, as well as between adjacent cell bodies,numerous gap junctions could be detected (Fig. 1D). Theirincidence was 5.5-fold higher on ECM than between cellsgrown on plastic. In cells cultured on plastic, numerous stressfibers were evident (Fig. 2A), while on ECM the number ofstress fibers was much reduced (Fig. 2B) and actin was oftenobserved in a diffuse pattern. In addition, actin staining wasdetected in the intercellular extensions of cells grown onECM. Cells grown on plastic displayed large vinculin-containing focal contacts at their ventral aspects (Fig. 2C),while cells plated on ECM had a low content of vinculin thatwas organized in thin focal contacts (Fig. 2D).

Levels of Vinculin, a-Actinin, and Actin Synthesis inGranulosa Cells Grown on ECM. Since changes in cellmorphology in response to the substrate on which the cellsare cultured were shown to affect both the organization andthe expression of cytoskeletal proteins (18), we followed thesynthesis of cytoskeletal proteins in granulosa cells culturedon plastic and on ECM. Cells were labeled for 3 hr with[35S]methionine, 48 hr after culturing on plastic and on ECM,and samples containing equal cpm of trichloroacetic acid-precipitable radioactive proteins were analyzed by high-resolution two-dimensional gel electrophoresis. The level ofsynthesis of several major cytoskeletal proteins was specif-

FIG. 1. Cell morphology andultrastructural characterization ofgranulosa cells grown on ECM.Granulosa cells were grown for 48 hron control (A and C) or on ECM-coated plastic (B and D). Live cellswere photographed after 48 hr of cul-ture (A and B), or the cells wereembedded in Epon (C and D) andsectioned for electron microscopicanalysis. The insert in D shows a gapjunction with the typical pentalaminarorganization. m, Mitochondrion; S,plastic surface. Large arrowheadspoint to actin filaments. The bracketsin D mark a gap junction; asterisksmark the ECM. (A, bar = 10 ,um; C,bar = 0.5 pan; Inset, bar = 0.1 ,Am.)

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FIG. 2. Organization of actin and vinculin in granulosa cellsgrown on plastic and on ECM. Granulosa cells grown on plastic (Aand C) or on ECM (B and D) for 48 hr were fluorescently stained todetect actin by rhodaminated phalloidin (A and B) orby a monoclonalantivinculin antibody and rhodamine-labeled goat antimouse IgG (Cand D). Arrowheads in B mark actin-containing cellular extensions.(Bar = 10 am.)

ically reduced in granulosa cells cultured on ECM as com-pared to cells grown on plastic (Fig. 3 A and B). Mostdramatic was the decrease (by a factor of 7-10) in thesynthesis of a 130-kDa protein, which we identified as theadhesion plaque protein vinculin. The vinculin spot comi-grated on two-dimensional gels with purified chicken gizzardvinculin (Fig. 3 C and D), and it could be immunoprecipitatedwith a monoclonal antivinculin antibody (34). In addition, adecrease by a factor of 2-3 in the synthesis of actin and ofa-actinin, both of which are involved in the formation ofadherens junctions (35), was also recorded in granulosa cellscultured on ECM (compare Fig. 3 A and B). In contrast, thesynthesis of a- and 3-tubulin did not change on ECM, asdetermined by the excision and counting of the radioactivespots from the two-dimensional gels (results not shown).Furthermore, when a Triton X-100 and high-salt-insolublefraction enriched in intermediate filaments was analyzed ontwo-dimensional gels, similar levels of the intermediatefilament protein vimentin were detected in both cultures (Fig.3 E and F). The Triton X-100-insoluble fraction contained=5% of total actin and vinculin in cells grown on plastic, butonly 1.5% of these proteins in cells cultured on ECM(compare Fig. 3 E and F). In addition, a-actinin, which is amajor protein of this subcellular fraction and which wasidentified by immunoblotting with anti-a-actinin antibody(Fig. 3 G and H), was found at a lower level (by a factor of2-3) in cells grown on ECM (compare Fig. 3 E and F). Atearly times after plating the cells (between 0 and 6 hr), thelevel of synthesis of vinculin, a-actinin, and actin was similaron ECM and on plastic, and the inhibition in their synthesiswas observed only 24 hr after cell seeding (Fig. 4; Table 1).

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FIG. 3. Vinculin, a-actinin, and actin synthesis in granulosa cellsgrown on ECM. Granulosa cells grown on plastic (A), and on ECMfor 48 hr (B) were labeled for 3 hr with [35S]methionine and analyzedby two-dimensional gel electrophoresis. (C and D) Coomassieblue-stained gels of cells grown on ECM. Purified chicken gizzardvinculin was added to D. A and B are total cell extracts, C andD areTriton X-100 and high-salt-soluble fractions. (E-H) Triton X-100 andhigh-salt-insoluble fractions enriched in intermediate filaments fromcells grown on plastic (E, G, and H) and on ECM (F). E and F arefrom 35S-labeled cells. (G) Coomassie blue-stained gel; (H) im-munoblot ofa gel identical to G, with a rabbit anti a-actinin antibody.a, Actin; t1, a-tubulin; t2, P-tubulin; arrowhead, vinculin; v,vimentin; a,, a-actinin.

Maximal effects on vinculin, a-actinin, and actin synthesiswere recorded between 48 and 72 hr after cell plating on thevarious substrata (Fig. 4 E and F).The Synthesis of Actin in Cells on ECM Is Reflected at the

Level of Actin mRNA. To follow the level at which thesynthesis of these cytoskeletal proteins are regulated, weprepared poly(A)+ and poly(A)- RNA from the cytoplasm ofgranulosa cells grown on either plastic or ECM. Similaramounts of RNA were analyzed by gel electrophoresis onagarose gels, and the RNA blots were hybridized with32P-labeled plasmid DNAs containing actin (Fig. 5, lanesA-D) and tubulin (lanes E and F) sequences. The amounts ofboth poly(A)+ and poly(A)- actin RNA were reduced signif-icantly in the cytoplasm of cells grown on ECM (lanes B andD) as compared to cells grown on plastic [lanes A and C, bya factor of4-6 in the poly(A)+ RNA fraction]. In contrast, thelevel of tubulin mRNA remained similar in cells grown onplastic and on ECM (only 30% reduction in cells grown onECM, lanes E and F). This is in agreement with the resultsobtained by the radiolabeling of proteins during their syn-thesis in the cells (Figs. 3 and 4).

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FIG. 5. Levels of actin mRNA inthe cytoplasm of granulosa cellsgrown on ECM. Poly(A)+ RNA(lanes A, B, E, and F) and poly(A)-RNA (lanes C and D) from granulosacells cultured on plastic (lanes A, C,and E) or onECM (lanes B, D, and F)for 48 hr were electrophoresed onagarose gels, transferred to nitrocel-lulose, and hybridized with 3P-labeled nick-translated plasmids con-taining actin (lanes A-D), or tubulinsequences (lanes E and F). kb,Kilobases.

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FIG. 4. Vinculin, a-actinin, and actin synthesis at various timesafter seeding on ECM and on plastic. Cells seeded on plastic (A, C,and E) and ECM (B, D, and F) were pulse-labeled for 3 hr with[35S]methionine starting at 3 hr (A and B), 21 hr (C and D), and 69 hr(E and F) in culture. Equal cpm of the Triton X-100-soluble fractionwere analyzed as described in Fig. 3. a, Actin; small arrowhead,vinculin; large arrowhead, a-actinin.

Relationship Among Morphological Changes, CytoskeletalProtein Synthesis, and Progesterone Production by GranulosaCells Grown on ECM. The morphological changes ingranulosa cells cultured on ECM are very similar to thosereported in these cells during their differentiation in responseto hormonal stimulation in vitro and during their normaldifferentiation in vivo (36-41). One parameter that is consid-ered to correlate with the differentiation of granulosa cells issteroidogenesis, which is characterized best by their produc-tion and secretion of progesterone (42). Therefore, we havesimultaneously followed the accumulation ofprogesterone inthe medium and the level of cytoskeletal protein synthesis ingranulosa cells cultured for various periods of time on eitherECM or plastic. The results summarized in Table 1 show thatcells grown on ECM produced high levels of progesterone,

Table 1. Cytoskeletal protein synthesis and progesteroneproduction in cells plated for different periods of time on ECM

Time of Vinculin, a-Actinin, Actin, Progesterone,incubation, % of % of % of % of

hr control control control control

3 115 97 107 1246 103 95 102 13924 42 70 85 34048 12 24 52 61172 14 20 38 2636

The relative rate of cytoskeletal protein synthesis was determinedby excising the radioactive spots from two-dimensional gels in whichequal cpm of total cell protein were analyzed after a 3-hr pulse atdifferent times after seeding the cells on plastic- or on ECM-coateddishes. Numbers represent the radioactivity on ECM-coated dishescompared to uncoated plastic dishes. Levels of progesterone accu-mulated in culture media were determined as specified in Materialsand Methods. The numbers represent amounts of the steroid thataccumulated on ECM-coated dishes compared to uncoated plasticdishes (which accumulated 22 ng of progesterone per 106 cells per 72hr). The results were obtained in two independent experiments withdifferent batches of cells. Progesterone production was determinedwith samples from triplicate plates and the differences did not exceed10%o. Data from the two experiments did not deviate by 20%/b from themean values presented in the table.

while cells grown on uncoated dishes produced only traces ofthis steroid. In addition, there was a parallel increase in theproduction of progesterone and an inhibition in the synthesisof cytoskeletal proteins, which are involved in the formationof cell contacts.An apparent increase in progesterone production was first

detected after 24 hr ofculture onECM (in correlation with thedecrease in the synthesis of cytoskeletal elements), whileafter 72 hr of culture, the levels ofprogesterone were 26 timeshigher in cultures grown on ECM-coated dishes compared tocultures on uncoated plastic dishes.To investigate the possible role of cAMP in mediating the

effect of ECM, we determined the level of cAMP at the timepoints shown in Table 1. We found that the level ofcAMP wassimilar in cultures on plastic and ECM and was in the rangeof 3.0-5.0 pmol per 106 cells.

DISCUSSIONThe present study demonstrated that the differentiation of ratgranulosa cells on ECM is associated with simultaneouschanges in the organization and expression of cytoskeletalproteins that are involved in the formation of cell contactsand in determining cell shape. In addition, numerous gapjunctions were detected between cells grown on ECM ascompared to cells grown on plastic where gap junctions weremore rarely observed. The EGTA/sucrose-dispersed freshlyprepared granulosa cells that were used for cell culturingdisplayed remnant gap junctions, most of them in the cyto-plasm in an annular form (unpublished observations). Severalcharacteristics of the morphological alterations induced byECM, such as changes in cell shape, formation of multilay-ered aggregates, reorganization of the microfilaments, andthe appearance of gap junctions, were also observed duringthe differentiation of granulosa cells in response to hormonalstimulation (37-42). This study shows that these changes inthe organization of the cytoskeletal elements in cells grownon ECM were followed by alterations in the synthesis of thecytoskeletal proteins vinculin, a-actinin, and actin. The factthat the cells on plastic produce more of these proteins couldbe interpreted as a stimulation of cytoskeletal protein syn-thesis by cells developing stress fibers (cf. ref. 43) or as adown-regulation in the cells grown on ECM. The firstpossibility is more likely, because [35S]methionine pulse-labeled freshly prepared granulosa cells display a low level ofsynthesis for these proteins (data not shown). The apparentdecrease in actin synthesis resulted from a decrease in thelevel of actinmRNA in the cytoplasm. On the other hand, thesynthesis of tubulins and of the intermediate filament proteinvimentin remained essentially unchanged during culturing onECM. These results are in line with previous studies thatdemonstrated that changes in cell shape and the extent ofcell-cell contact are reflected at the level of organization andexpression of the respective cytoskeletal proteins (refs. 31and 43-45; for reviews, see refs. 18 and 19). Whether such

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changes are obligatory for the differentiation of granulosacells in vivo is not yet known. However, it should be notedthat the most highly differentiated layers of granulosa cells inthe preovulatory follicle are adjacent to the basement mem-brane, have a columnar shape, and display a much highercontent of receptors to luteinizing hormone and a highersteroidogenic activity (26, 27). Therefore, this study supportsthe hypothesis that the proximity of granulosa cells to thebasement membrane is important in determining a gradient ofdifferentiating cells with the less differentiated cell layersbeing more distal from the basal lamina and closer to thecenter of the follicle. In addition, granulosa cells cultured insuspension as multicellular aggregates on nonadhesivepoly(HEMA)-coated dishes (1) exhibit a similar decrease inthe synthesis of these cytoskeletal proteins together with anincrease in progesterone production (data not shown), sug-gesting that the differentiation of these cells and the changesin cell shape and cytoskeletal gene expression are linked.Furthermore, our preliminary results with granulosa cellsstimulated to differentiate by gonadotropin and cAMP alsoshow a decrease in the synthesis of vinculin, a-actinin, andactin as compared to unstimulated cells. Whether the stim-ulation of granulosa cell differentiation and the associatedalterations in cytoskeletal protein expression induced byECM are mediated via cAMP, as is the case with gonadotro-pins (46), is unclear, since cell cultures on plastic and onECMhad similar levels of cAMP. This does not rule out thepossibility of short and transient increases in the level ofcAMP in cells grown on ECM. An alternative mechanism bywhich ECM may affect granulosa cells could operate viachanges in the organization of cell membrane-associatedproteoglycans (such as heparan sulfate) in response to theirinteraction with the ECM. Granulosa cells synthesize andsecrete proteoglycans (47), and it was suggested thattransmembrane proteoglycans may interact with cytoskeletalelements that terminate at the plasma membrane (48, 49).Changes in cell shape were shown to be necessary for the

expression of the differentiated phenotype in chondrocytes(3, 4), erythroid cells (2), hepatocytes (50), and during thedifferentiation of embryonal corneal epithelium (9). Theculturing of corneal epithelium on ECM was shown to befollowed by a reorganization of the epithelial cytoskeleton(24, 25). In addition, chicken heart fibroblasts seeded ongelatin-coated substrata formed mainly contacts of the"ECM type," which contained a-actinin, but not vinculin(35).Changes in cell shape and alterations in cytoskeletal

protein gene expression were also shown to precede and tobe necessary for the adipocytic differentiation of3T3 cells (5,6). In 3T3 adipocytes, the conversion from a fibroblastic to aspherical shape was followed by a dramatic decrease in actin,tubulin, and vimentin synthesis at a very early step in thedifferentiation ofthese cells, before the induction oflipogenicenzymes (5). When these cells were cultured on fibronectin-coated dishes, the alterations in cell morphology and theinduction of lipogenic enzymes were all prevented (6).Taken together, these studies strongly support the hypoth-

esis that changes in cell contacts and shape and the changesin cytoskeletal gene expression are programmed alterationsthat may be involved in the regulation of the subsequentdifferentiation process. The extracellular matrix organized asthe basal lamina, which determines cell morphology in vivo,may play an important role in these events.

We thank Ms. Tamar Kreizman, Mr. A. Almoznino, and Mr. S.Himmelhoch for expert technical assistance; Drs. I. Ginzburg and M.Shani for providing cDNA clones; Dr. B. Geiger for fluorescentantibodies; Dr. I. Vlodavsky for the ECM-coated plates (some ofwhich were kindly provided by International Biotechnologies, Kiryat

Hadassah, Jerusalem); and Dr. F. Kohen for helpful discussions. Wethank Mrs. M. Kopelowitz and R. Levin for excellent secretarialassistance. This study was supported in part by a grant from theIsrael-USA Binational Foundation (Jerusalem) to A.B.-Z., and fromthe Rockefeller Foundation (New York). A.A. is the incumbent ofthe Joyce and Ben B. Eisenberg Professorial Chair in MolecularEndocrinology and Cancer Research.

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