Isolation by Fluorescence-activated Cell Sorting of Chinese Hamster ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY It:)

Vol. 266, No. 18. Issue of June 25, pp. 11746-11752,1991 Printed in U.S.A.

1991 by The American Society far Biochemistry and Molecular Biolow, Inc

Isolation by Fluorescence-activated Cell Sorting ofChinese HamsterOvaryCell Lines with Pleiotropic, Temperatureconditional Defects in ReceptorRecycling* (Received for publication, December 27, 1990)

Cynthia Corley Cain$, Russell B. WilsonB, and RobertF. Murphyll From the Department of Biological Sciences and the Center forFluorescence Research in Biomedical Sciences, Carnegie Mellon Uniuersity, Pittsburgh, Pennsylvania 15213

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT 1.11, was selected for further characterization. In TfT1.ll the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 “C the surface binding of transferrin is reduced to 18%of parental cells at 41 “C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditiona15.5-fold decrease in the initial rate of transferrin recycling. TfT 1.11 cells alsorapidly lose 89%of their ability to take up a2-macroglobulinafter the temperature shift to 41 “C. These data indicate that the TfT 1.1 1 cell line has a pleiotropic defect in receptor recycling.

from receptoris coupled to endocytic acidification (forreview, see 10). Segregation of most receptor-ligandcomplexes occurs in the endosome, an early, prelysosomal endocytic compartment with a low buoyant density (11-15). The pH of early endosomes is regulated in many cell types to approximately pH 6 (16-18), a pH sufficient to drive the dissociation of many ligands and receptors. This allows recycling to occur before exposure to the degradative environmentof the lysosomes.Onceligandshavedissociatedfrom their receptors, segregation largely becomes a problem of the relative retention of fluid during therecycling of membrane and membranebound material (19). Although the mechanismfor dissociation of many receptors and ligands has been determined, very little is known about the mechanism forsegregation and recycling. It has been suggested that the morphology of the endosome may be responsible for the segregation of receptors and ligands (20). Although the relativesurface area to volume ratios of the recycling vesicles and endosomes may account for the segregation of free ligand fromreceptor duringrecycling (21), there are several membrane-bound proteins, including epidermal growth factor receptor,IgG Fc domain receptor, and mannose Several early observations indicated that there is extensive 6-phosphate receptor, which are retained and carried further recycling of internalized membrane and protein to the cell in the pathway (11, 15, 22, 23). This suggests that there is a surface after endocytosis (reviewed in Refs. 1 and 2). During specific mechanismtosortmembrane-boundcomponents extended periods of endocytic activity cells internalize a large from each other in addition to a means of separating mempercentage of their plasma membrane and a large volume of brane from fluid. Sorting for selective internalization is acfluid although the dimensions of the cells do not change. In complished attheplasmamembranethrough cytoplasmic addition, for many molecules thetotalamount of ligand determinants on membrane proteins that direct their binding internalized greatly exceeds the number of cell surface recep- to adaptor proteins (adaptins) in clathrin-coated regions of tors. Cells are able to sustain continuous uptakeof ligand for the plasma membrane (24). Although a similar mechanism long periods of time even in the absenceof protein synthesis couldplay a role in receptor recycling, it is currently not (3-5). The kinetics of ligand accumulation indicate that recep- known if recycling is a directed process (a recycling signal is tor recycling is very efficient and that a single receptor can needed) or a default pathway (a retention signal is needed). be internalized hundredsof times. It is clear, however, that the rateof receptor recycling can be To accumulate ligand and allow for receptor recycling cells regulated in both a growth state- and cell-type-dependent must have a mechanism to segregate material to be recycled manner (25-30). For manyreceptorsaffinity for from that to be retained. To begin to identify molecules involved in this process we ligand is low at acidic pH (6-9), and dissociation of ligand selected cell lines defective in receptorrecycling using labeled transferrin (Tf)’ asa marker for its receptor. Tf, a major iron * This work was supported in partby National Institutesof Health transport protein, binds with high affinity tosurface receptors Grant GM32508 andNational Science FoundationGrantDCB8903657 (to R. F. M.). The costs of publication of this article were and is internalizedduring endocytosis. Unlikemostother ligands, however, Tf escapes lysosomal degradation by recydefrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 cling back to the cell surface bound to itsreceptor (31, 32).A U.S.C. Section 1734 solely to indicate this fact. single cycle of internalizationand recycling is completed

Supported by an American predoctoral fellowship of the American Association of University Women Educational Foundation. 5 LawtonChilesBiotechnology Postdoctoral Fellow of the National Institutes of Health, GM13179. ll To whom correspondence should be sent: Dept. of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, P A 15213. Tel.: 412-268-3480; Fax: 412-268-6571.

‘ T h e abbreviations used are: Tf, transferrin;TfR,transferrin receptor; a2M,a,-macroglobulin; a-MEM, minimal essential medium, a-modification; Cy5, cyanine5.18-OSu: FITC, fluorescein isothiocyanate;MES, 2-(A”morpho1ino)ethanesulfonic acid CHO, Chinese hamster ovary.

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CHO Cell Lines with Pleiotropic Receptor Recycling Defects within 10-15 min (4,5 , 16). To isolate recycling defective mutants cells were selected for retention of labeled Tf after a chase with excess unlabeled Tf. In this paperwe describe the isolation of.these cell lines and the initial characterizationof the clone TfI'l.ll, whichshowsa temperature-dependent, pleiotropic defect in receptor recycling. EXPERIMENTALPROCEDURES

Tissue culture supplies were purchased from GIBCO, and other materials were purchased from Sigma, unless otherwise noted. Preparation of a,-Macroglobulin-a2-Macroglobulin (a2M) was prepared from freshhumanserum. A 7-26% polyethylene glycol fraction (33) was dialyzed against distilled water, centrifuged (40,000 X g), anddialyzed against buffer A (0.15 M NaCl, 0.02 M NaPO,, pH 6.0). The solution was thenfractionated by zinc affinity column chromatography (34). The a,M bound to the affinity column (2.5 X 27 cm) was eluted as described (35) except that 50 mM acetate, pH 4.5, was used for elution. The eluted proteinwas then dialyzed against buffer A and fractionated by gel permeation on a Sephacryl S-300 column (2.5 X 90 cm). The purified asM was then activated with methylamine as described (33). Synthesis of Fluorescent Conjugates-FITC-dextran (70,000 daltons) was prepared by thedibutyltin-dilauratemethodwiththe modifications described previously to reduce free dye contamination (36). Tf (human diferric, Miles Scientific, Naperville, IL) was labeled with Cy5.18-OSu (Cy5) (52) using the protocol described previously for Tf conjugated with lissamine rhodamine sulfonyl chloride (16). Cy5.18-OSu has properties similar to those of Cy5.12-OSu (37) and was a generous gift from Dr. Alan Waggoner, Carnegie Mellon University. The resulting dye to protein ratioswere 2.75 for the conjugate used in mutant isolation experiments and 5.0 for the conjugate used in the characterization experiments. aeM was labeled with Cy5, with a resulting dye to protein ratio of 7.1. All of the Cy5 protein conjugates had binding specificities of greater than 90%. Typical ratios of Cy5 fluorescence to background fluorescence from unlabeled cells were 130:l for Tf (10 pg/ml) and 45:l for n2M (10 pg/ml). Flow Cytometry-A dual laser FACS 440 flow cytometer (Becton Dickinson ImmunocytometrySystems) equipped withargonand krypton lasers was used for all analyses. FITC fluorescence (488 nm excitation, 400 milliwatts) was collected using a 530 nm band pass filter (30 nm band width), and Cy5 fluorescence (647 nm excitation, 200 milliwatts) was collected using a 670 nm band pass filter (13.5 nm band width). A FACStar''."' equipped with argon and heliumneon lasers was used for sorting. Isolation of Recycling Defective Cell Lines-CHO WTB cells (obtained from Dr. BrianStorrie, Virginia Polytechnic Institute and State University)were maintained a t 33 "C in a-MEM supplemented with 10% calf serum (HyClone Laboratories, Logan, UT),100 units/ ml penicillin, 100 pg/ml streptomycin, 0.29 g/liter L-glutamine, and 4 g/liter proline (c-a-MEM). Cells were mutagenized by incubation in c-n-MEM with 250 gg/ml ethylmethane sulfonate for 24 h and allowed to recover for 2 weeks prior to the first sort. Cells were incubated a t 41 "C for 0.5, 1, 1.5, or 2 h and labeled for 30 min with 2.5 pg/ml Cy5-Tf in a-MEM. The cells were then incubated for 1 h in c-a-MEM with 1 mg/ml unlabeled human diferric Tf and 2 mg/ ml FITC-dextran a t 4 "C (to determine the init,ial amount of Tf internalized) or a t 41 "C (for samples to be sorted). After labeling, the cells were washed six times with ice-cold a-MEM salts (116 mM NaCI, 5.4 mM KC1, 0.2 mM CaCI,, 0.8 mM MgSO,, 10 mM NaHaP04, pH 7.4) andsuspended by scrapingintoa-MEM.Samples were maintained on ice during analysis andsorting. The 0.1% of the cells which showedthe highest Cy5-Tf and FITCdextran labeling were sorted from samples from each incubation time (1,000 cells sorted). Parallel sorts using less stringent criteria (1%of the cells) were unsuccessful. Cells were sorted into 4 ml of ice-cold cWMEM supplemented with additional antibiotics (0.1% gentamicin, 1%fungizone, and 0.1% nystatin) and plated at 33 "C. After 2 days the sorted cells were maintained in c-a-MEM without additional antibiotics. Each sort was maintained as a separate population (four incubationtimes),andeachpopulation was expandedtoobtain sufficient cells for a second sort (19 days). Each population was sorted a second time (same expression time and sort criteria), expanded, and analyzed. After expansion of the second sort, a distinct doublepositive population was present in the2-h population andwas sorted.

11747

The Tff cell lines were clonedfrom thisthirdsort by limiting dilution. Transferrin Binding-Cells were plated in 12-well tissue culture plates(Corning Glassworks, Corning, NY) 2 daysinadvance for approximately 80% confluence at the time of use. The plates were shifted to 41 "C for various times andwashed with a-MEM salts. The cells were then labeled for a t least 1 h a t 4 "C in a-MEM containing Cy5-Tf at the indicated concentrations. The cells were washed six times with a-MEM salts, suspended by scraping into a-MEM salts, and analyzed immediately (within 30-60 s after removal of TO. For the determination of TfR affinity, cells were incubated in aMEM for 30 min at 4 "C, washed two times with a-MEM salts, and labeled for 1 h at 4 "C in a-MEM containing 1 mg/ml bovine serum albumin and various concentrations of Cy5-Tf (2-200 pg/ml, three samples/concentration). The samples were then washed and analyzed as above. Mean fluorescence values were corrected for nonspecific binding, determined a t each concentration by competition using 5 mg/ml unlabeled Tf. For Scatchard analysis the concentrationof Tf added was used as an estimate of free Tf (since the fraction of input Tf bound to cells was negligible under the conditionsused). Transferrin Ezternalization-Cells were plated as described for Tf binding experiments. Samples of T f f l . l l a n d W T Bwere shifted to 41 "C for 3h or maintained a t 33 "C, washed with a-MEM, and incubated for 30 min at the appropriate temperature in0.5 ml of N MEM containing 1 mg/ml bovine serum albumin and 10 pg/ml Cy5Tf. Thecells were then chilled quickly to 4 "C by washing twice with ice-cold a-MEM salts. To remove surface-bound Tf cells were incubated at 4 "C in 0.5 ml of stripping buffer (150 mM NaCI, 100 p M desferrioxamine mesylate (Ciba-Geigy), 50 mM MES (Research Organics, Cleveland, OH), pH 4.5, for 5 min followed by two successive 5-min incubations in n-MEM salts containing100 p~ desferrioxamine mesylate. The cells were then warmed quickly to the appropriate temperature by the addition of 2 ml of warm c-a-MEM with 0.5 mg/ ml unlabeled Tf. After various times the cells were againchilled quickly to 4 "C with two washes of ice-cold a-MEM salts. The cells were maintained a t 4 "C until all samples had been collected, washed five times witha-MEM salts, and suspended by scraping immediately prior to analysis by flow cytometry. The data were fit by one or two exponentials using a nonlinear least squares fitting program(38). Nonlinear fits to all data were made using four models: a single exponential (two free parameters), a single exponential with a nonrecycled component (three free parameters), two exponentials (four free parameters), andtwo exponentials witha nonrecycledcomponent (five free parameters). The root mean square error (normalized toa percent error by division by the mean Y value for each data set)was used to judgegoodness of fit. For the 33 "C data the first model yielded significantly higher error values (11.6 and 16.8% for WTB and TfTl.11,respectively) than the other three. Since the values error for these three models were within 0.03% of each other, the second model (single exponential witha nonrecycled component) was chosen (error values were 6.5 and 6.8% for W T B a n d T f f l . l l respectively). , For the41 "C data, the first two models gave higher error values (21.2 and 14.5% for WTB, 12.9 and 10.2% for TfTl.ll) than the last two (9.6-9.7% for both WTB and TfTl.ll). The decrease in error of less than 0.1% between the third and fourth models was not considered to justify the addition of a free parameter, so the third model was chosen. Results are also shown for the second model for TfTI.ll at 41 "C since the difference in error between the second and third models was only 0.6%. To determine how similar the pathways in TfT 1.11and WTBwere at 41 "C, an additional fitwas made using the third model with the rate constants fixed to those obtained withWTB (themodel thus has only two free parameters). The error obtained (14.2%) was significantly larger than the others for this data set (including the first model, whichalso had only two free parameters); the results are shown in Table IV for comparison only. For all of the samples there was a significant but variable fraction of the Tf which was released very rapidly from the cells (tIr2< 30 s). This may represent the "fast" external compartment described by McKinley and Wiley (39) for adherent cells, which is caused by trapping of ligand between the cells and the plates at low temperatures. No attempt at fitting this component was made since the halftime of release was shorter than the first point (30 s). To estimate the amount of cell-associated Tf excluding this external compartment, the nonlinear fits were extrapolated to 0 min. The raw data were then converted toa percentage of this value. a2-Macroglobulin Uptake-Cells were plated as described for the Tf binding experiments, shifted 41 to "C for varioustimes, andlabeled

11748

CHO Cell Lines with Pleiotropic Receptor

for 6 min at 41 "C in a-MEM with 10 pg/mlCy5-asM.Toclear receptors of unlabeled a2M from the serum, the cells were incubated without serum at either 33 or 41 "C for 30 min prior to labeling. After labeling the cells were chilled quickly to 4 "C, washed twice with aMEM salts, suspended by scraping into a-MEM salts, and analyzed by flow cytometry.Short term uptakewas measuredinstead of surface binding because very little binding could be detected at 4 "C even after a 1-h incubation. The temperature dependence of a2Mbinding has been noted for other cell types as well (40). Measurements of cell-associated a s M reflect both surface bound and internalized ligand because the off rate, like the on rate, is quite slow. Cellular ATP Leuels-Cellswere plated as for the Tfbinding experiments. The cells were shifted to 41 "C for 4 h or maintained at 33 "C prior to harvesting by scraping at 4 "C. The amount of ATP/ sample was determined using a luciferin/luciferase ATP determination kit for somatic cells (Sigma) and a Thorn EM1 PMT (800 V) coupled to a Thorn EM1 photon counter (Thorn EM1 Gencom Inc., Fairfield, NJ). The average number of cells/sample was determined from parallel samples. Protein Synthesis-Cells were plated as for the Tf binding experiments. The plates were shifted to 41 "C for 4 h or maintained at 33 "C, washed with a-MEM salts, and incubated at the appropriate temperature for 15 min in assay medium (deficient Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 units/ ml penicillin, 100 pg/ml streptomycin,0.292 g/liter L-glutamine, 0.105 g/liter L-leucine, 0.146 g/liter L-lysine, 4 g/liter proline, and0.3 mg/ liter L-methionine). 8 pCiof Tran35S-label(ICN; Irvine, CA) was added to half of the wells, and the incubation continuedfor 45 min. The cells were then washed three times with a-MEM salts. The cells that did not receive radioactivity were suspendedtrypsin by treatment and counted with a Coulter Counter (Coulter Corp; Hialeah, FL). The cells receiving radioactivity were lysed in85 pl of lysing solution containing0.1% sodium dodecylsulfate,0.1 mg/ml deoxyribonuclease I, 1 mM CaCI2,and 1 mM MgC12. The lysatefromeachwellwas transferred to 2.5-cm squares of Whatman 3MM, soaked for 20 min in 5% trichloroacetic acid containing 0.5 mg/ml L-methionine, and washed once in 95% ethanol. The filter papers were dried, and the radioactivitywasmeasuredin a liquid scintillation counter using Ecolume (ICN) as the scintillant.

Recycling Defects

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FIG. 1. Conditions for isolation of recycling mutants. Mutagenized WTB cells were incubated for 2 h at 41 "C and labeled for 30 min with Cy5-Tf at 41 "C ( A ) . The labeled Tf was then chased from the cells during a 1-h incubation with excess unlabeled Tf and FITC-dextran (C). Veryfewcellswere detected as double-positive mutants were selected events (quadrant IZ) prior to sorting. Recycling as those cells labeled with both Cy5 and FITC. The population of cellsobtainedafter two sorts waslabeled ( B ) andchased (D)as described for WTB cells. A clear double-positive population, comprising approximately 9% of the total cells, was observed (arrow). Contours were drawn at 5, 10,15, and 20 events/bin (5,000 events total).

there was an enrichmentof large cells (asjudged by increased light scattering) which had a concomitant increase in the number of receptors/cell (data not shown). In the larger cells, the Cy5 fluorescence after chasewas higherthan in the control cells (Fig. 1, C and D, quadrant I V ) , which accounts for their selection; however, the higher fluorescence represented the same percent of the internalized Tf as in the unselected RESULTS population (not shown). The apparent increase is caused by Cell Sorting-To isolate cells with temperature-conditional an increase in the total number of receptors rather than a defects in receptor recycling we explored the feasibility of difference in receptor recycling. In addition to thelarge cells using fluorescence-activated cell sorting t o select cells that present after thesecond sort there was a distinct subpopulawere unable t o recycle labeled Tf administered aftera shift to tion of double-positive cells in the 2-h sample, comprising a higher temperature. A continuous incubation in the presence approximately 9% of the total cells observed (Fig. lD, quadof labeled Tf during the temperature shift could not be used rant 11).This subpopulationwas sorted andcloned bylimiting t o label the cells since normal WTB cells were observed t o dilution. retain some labeledtransferrin afterchase. The accumulation Failure torecycle Tf (andTfR) would be expectedt o result of labeled Tf occurred at a rate of approximately 5% of the in a decrease in cell surface TfR. If this is the case, expression total internal Tf for each h of labeling and was inhibited by time(time at 41 "C) would be a criticalvariableinthis the additionof excess unlabeled Tf during the pulse (data not shown). This phenomenon was observed for both Cy5- and selection protocol. At early times no Tf would be trapped FITC-Tf and was also observed in CHO K1 and Swiss 3T3 because of the lack of expression of the defect whereas at late of surface TfR would be decreased, reducing cells (data not shown). The retention of a small fraction of times the number initial Cy5-Tf labeling and decreasing the potential signal. the internal Tf has beendescribed in other cell lines as well (27, 41). To avoid this problem cells were incubated at 41 "C This effect was demonstrated by examining the kinetics of for various times toallow expression of heat-sensitive lesions, expression of the trapping phenotype for the population of sort (Fig. 2). The pulse labeled with Cy5-Tf for 30min, and chasedfor 1h with double-positive cellsobtained after the third excess unlabeled Tf. Cy5 was used as the fluorescent marker trapping reaches a maximum at 3.5 h and then decreases. because it gave very high signalto noise ratios (typically 1 3 0 1 After 5 h at 41 "C, the uptakeof Tf during the30-min pulse for the initial labeling: see "Experimental Procedures"). T o is only 35% of the uptakeat 33 "C. Although there appears to at to select for T f trapping mutants, ensure that the sorted cells were capable of internalization, be an optimal timewhich may be differentfor each recycling mutant FITC-dextran was included in the chase media as a marker the expression time of fluid phase endocytosis. The desired mutantswere selected isolated. Initial Characterization of Clones-To verify that theclones t o be positive for Cy5-Tf (caused by failure t o recycle it during hadtemperature-conditional defects in receptor recycling the chase) and also positive for FITC-dextran (normal for clones were screened for temperature-dependent loss of surinternalization). Examples of the sort conditions are shown shown in TableI. Since theseclones in Fig. 1. Double-positive cells were sorted from the samples face TfR. The results are a bulk were derived from a single mutagenesis and carried as chased at 41 "C (Fig. IC, quadrant 11). population for several generations after sorting it is possible There was no detectable enrichment for double positives after the first sort (data not shown). After the second sort that they may be derived from a single mutation. However,

CHO Cell Lines with Pleiotropic Receptor Recycling Defects

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FIG. 4. Trapping of Tf in TfT 1.11 is temperature conditional. WTB and TfTl.llcells were labeled and chased asdescribed in Fig. 1 after a 2-h incubation a t either 41 "C ( B ) or 33 "C (C). Histograms of side (90 ") light scatter (a measureof cell size) are also shown ( A ) .

I . I . I . I . , . , I

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TABLEI1 Effect of incubation temperature on ATP levels and proteinsynthesis in WTB and TfTl.ll Data are meansf S.D. of triplicate samples. Temperature ATP levels 35Sincorporated

Time at 41 C (h) FIG. 2. Kinetics of expression of the Tf trapping phenotype. WTB cells (0)and cells sorted from the double-positive population cpmlcell "C shown in Fig. 1D (A)were incubated at 41 "Cfor the indicated times, W T B labeledwithCy5-Tf, andchasedasin Fig. 1. The fluorescence 33 remaining cell associated after the chase is expressed aaspercentage 41 of the amount taken up during the pulse of (means duplicate samples). Relative change with temperature shift TABLEI Screening of TfT clones for loss of surface transferrin receptors TfT1.ll 33 Cells were labeled a t 4 "C after a 4-h incubation a t 41 or at 33 "C. 41 Values are normalized to WTB values at each temperature. Relative change with temRelative Tf Binding perature shift WTB Tff1.2 TfTl.ll TfT1.13 TfT1.14 TfT 0.241.16

33 "C

41 "C

1.00 0.68 1.02 0.93 0.99 0.90

1.00 0.18 0.28 0.26 0.29

pglcell

*

808 62 563 k 72 0.7 k 0.1

104.5 f 10.7 197.2 f 16.3 1.9 f 0.2

709 k 62 696 2 96 1.0 f 0.2

182.0 f 22.1 304.8 & 16.8 1.7 2 0.2

reduction in the amountof Tf internalized in a 30-min pulse (compare Fig. 3, A and B), and themajority of the cells are clearly positive for both dextran accumulation and retention of Cy5-Tf (Fig. 30). However, TfT1.11 shows some decrease in the extent of dextran labeling (Fig. 30). This defect in dextran accumulation is not caused by adifferencein the initial rate of dextran internalization but by a difference in the efflux of fluid after endocytosis.' The retentionof Cy5-Tf in TfT1.ll is temperature dependent, as there is a significant amount of Tf retained after chase at 41 "C(Fig. 4B) but little trapping at 33 "C(Fig. 4C).TfI'l.ll is similar insize to WTB in contrast to the majority of the cells after the (Fig. a), second sort (seeabove). The phenotype of TfT1.11 is not caused by a gross alteration in cellular metabolism. There is no significant decrease in the levels of ATP in TfI'l.ll cells and no significant difference in the relative rates of protein synthesis after temperature shift (Table11). Loss of Surface Transferrin Receptors-Fig. 5 shows the kinetics of loss of apparent cell surface Tf binding sitesfrom TfT1.11cells during incubationat 41 "C. Within 4 h after the 1 I I shift to 41 "C the bindingdecreased to less than 30% of the 2 3 24 3 4 initial binding andless than 25% of the increased binding to Log FlTC Fluorescence WTB at the elevated temperature.The loss of receptors FIG. 3. T f T l . l l cells trap Tf. WTB (A and C) a n d T f f l . l l ( B occurred with a t1r2 of approximately 2 h and began almost and D ) cells were incubated for 2 h a t 41 "C, labeled for 30 min with immediately after shifting the temperature. Scatchard analyCy5-Tf (A and B ) , and chased for 1 h with FITC-dextran andexcess sis (Table 111) indicates that the loss of binding is caused by unlabeled Tf ( C and D )as in Fig. 1. Contours are drawn at 20,40,60, a decrease in the number of surface receptors rather than a 80, and 100 eventshin (20,000 events total). change in receptor affinity. After a 4-h incubation at 41 "C there was no difference in the TfR affinity of WTB and one of the clones, TfT1.2, appears to be different from the TfT1.11cells; the calculatedKd for WTB isin good agreement others, as itshows a 30% reduction inTf binding (relative to with values reported previously (42). Under these conditions parental) even at the permissive temperature.The clone the number of surface receptors in TfTl.11is reduced to 18% TfT1.11 was selected because it had normal levels of TfR at of WTB. the permissive temperature and showed a significant loss of TfR at thenonpermissive temperature. ' R. B. Wilson, C. C . Cain,and R. F. Murphy,manuscriptin After a 2-h incubation at 41 "C, TfTl.11 showsa small preparation.


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FIG. 5. T f T l . l l cells lose cell surface TfFt at 41 "C. WTB (0)and TfTl.ll (A)cells were incubated for the indicated times at 41 "C and labeled with Cy5-Tf at 4 "C. Immediately prior to analysis the cellswerewashedandsuspendedbyscraping.Shown are the meansand standard deviationsfortriplicatesamplesfromthree experiments ( n = 9) normalized to the WTB 33 "C average value.

FIG. 6. TfR are lost from the cell surface because of a defect affecting the rate of recycling. WTB (0)and Tfl'l.ll (A) cells were labeled with Cy5-Tf for 30 min either at 33 "C ( A ) or at 4 1 "C after incubation at 41 "C for 3 h (B).Cells were chilled to 4 "C and surface Tf removed by incubation at low pH (pH 4.5). The cells were warmedrapidly in the presence of excessunlabeledTf to allow recycling and werechilled to 4 "C at the indicatedtimes to halt externalization. Datashown are mean fluorescence values andstandard deviations for triplicate samples from three experiments( n = 9) normalized to the calculated internal Tf at t = 0 (see "Experimental Procedures").Curues are single exponentials fit to the individualdata points for the 33 "C samples and two exponentials fit to the 41 "C data (see Table IV).

TABLEI11 Transferrin receptor properties of W T B and TfTl.1 I TABLEIV Scatchard analysiswas performed on Cy5-Tf binding data obtained Kinetic constants from exponential fits of the Tf recychg data as described under "Experimental Procedures." Means andstandard deviations from two experiments areshown. The estimated errors in The data in Fig. 6 were analyzedas described under "Experimental the linear fit parameters for each experimentwere propagated during Procedures." For 33 "C samples, first-order rate constants were estiaveraging to yield the estimated errorof the average. mated from nonlinear least squares single-exponential fits of the data (model 2), allowing fora component that is not recycled fromthe cell Relative no. of K d (t,,>> 90 min). For the 41 "C samples, the slow component was fit by receptors/cell" a second exponential (assumes that all of the Tf is recycled, model flM 3). Results of two alternative fits for TfT 1.11at 41 "C are also shown. WTB 18.6 f 3.6 1.00 f 0.03 Fast component

TfTl.11 18.5 f 3.5 0.18 f 0.01 " Cy5-Tf fluorescence was normalized such that the relative number of receptors/WTB cell was one in each experiment.

Model" Initialrateb Fraction

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Slow component

Fraction

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%of initial

rnin

33 "C WTB >90 2 8.5 8.37.6 91.5 >90 Tffl.11 18.5 8.5 2 81.4 6.6 Kinetics of Transferrin Externalization-Receptor loss from the cell surface couldbe the resultof either an increase in the 41 "C rate of receptor internalization or a decrease in the rate of WTB 3 24.2 62.0 1.8 27.5 38.0 of clearreceptor recycling. To address this point the kinetics TfTl.ll 3 66.6 62.64.4 5.9 37.4 ance of a 30-min pulse of Cy5-Tf were measured for 13.8 WTB 68.7 2 3.5 TfTl.11 Tffl.11 6.3' 2.5 27.5 1.8 93.7' and TfTl.11 at permissive and nonpermissive temperatures. At 33 "C the kinetics of loss of labeled Tf were similar for See "Experimental Procedures" for descriptionsof models. W T B a n d T f T l . l lalthough the fraction of Tf which is not Calculated as the product of the rate constant (ln(2)/t,) and the recycled (at leastover 90 min) is higher in Tffl.ll (Fig. 6A). fraction of Tf which is recycledthrough the fast (or only) component. Model 3 but with the rate constants fixed to those obtained for First-order rate constants and maximum fraction lost were estimated by nonlinear least squares fittingof a single expo- the WTB 41 "C data. nential (Table IV). The products of these values (the initial rate) were similar for WTB andTffl.ll. At 41 "C the loss of only a single component is present a t 41 "C in TfT1.ll with Tf is accelerated in WTB whereas it is slowed in TfTl.11 a rate more than seven times slower than WTB. Regardless (Fig. 6B). When cells are shifted to 41 "C the initial rateof of which model is used, the analysis leads to theconclusion recycling increases by a factor of 3 in the WTB cells, but a that the TfTl.ll mutant retains Tf and TfR at the nonpersignificant fractionof the Tfis cleared from the cells through missive temperature because of an alteration in the rate of a slow pathway (tllz= 27 min). In TfTl.11 cells recycling receptor externalization. Reduced az-Macroglobulin Uptake-To determine whether or altered dramatithrough the fast pathway is either absent cally. When the data are using fit the samemodel as for WTB the Tf recycling defect in TfTl.11 is caused by a general the results suggest that a shift to 41"C redirects the bulk of defect in therecycling pathway, aZMaccumulation was measthe Tf through the slow component (63% in TfT1.ll cells ured for TfTl.ll and WTBcells after the temperature shift. Cells were incubated for various times at 41 "C and labeled comparedwith 38% in WTB cells). Inaddition,therate constants for clearance through both the fast andslow com- with Cy5-cupMfor 6 min at 41"C. Differences in azM uptake to differences ponents are 2.5-3 times slower in TfTl.11 cells than in WTB in these short incubations are presumed reflect cells at 41 "C. Since the errorvalues obtained when the data in the number of surface receptors, as there is no difference are fit with themodel used for the 33"C data areonly slightly between the two cell lines in the amountof dextran internalhigher than for the two-component model, it is possible that ized in a 6-min pulse a t either 33 or 41 "C.' Interestingly,


11751

T f T l . l l cells have 1.4 times as many a2M receptors as WTB TfT1.ll, thiscell line shows a reduction in Tfsurface binding cells at 33 "C (Fig. 7). a2M uptake is lost rapidly after the which is caused by a shunting of 25% of the TfR to a pathway temperature shift (t1,2 of approximately 1.5 h) and declines with a very slow rate of return to thecell surface ( t1/2greater by greater than90% by 4 h. The loss of surface azM receptor than 100 min).However, the loss of TfR from the cell surface indicates that the defect in TfT1.11is notspecific forthe TfR in AF192 is not as severe as the loss in TfI'l.11 at 41 "C (a 25% reduction compared with a 82% reduction in T f T l . l l but is a pleiotropic defect in receptor recycling. after the temperature shift). Like AF192 cells, TfTl.11 cells DISCUSSION at 41 "C show anincreaseintheamount of Tf released through a slow pathway. Unlike AF192, however, there isalso Mostendocytosis-defective mutants describedpreviously a 2.5-3-fold difference in the rate constants for recycling from have been isolated by selection against some aspect of the TfI'l.11 (at 41 "C both the fast and the slow pathways in endocytic pathway (16, 43-46). Inall of these selection there is a fraction of Tf which recycles slowly in WTB cells schemes mutant cells were isolated by killing normal cells. as well). When the TfT1.11 data are fit using the WTB rate We have isolated cell lines with temperature-conditional defects in receptor recycling during endocytosis using the trap- constants, 94% of the Tf appears torecycle through the slow pathway (Table IV). It is possible that these two cell lines ping of labeled Tf as a marker selectable by fluorescencehave the same defect, with the difference in severity caused activated cell sorting. There are several advantages of using by differences between temperature-conditional and nonconcell sorting forselection of mutants. Positive selection by ditional alleles. However, it was not determined if the defect sorting allows the isolation of new classes of mutants which in AF192 was specific forTf or affected other ligands as well. would be difficult to isolate usingnegativeselection techThere is considerable evidence that intrinsic properties of niques. Mutants are isolated under conditions that are not receptors affect recycling. A point mutation in the cytoplasmic lethal, and the selection does not require the perturbationof domain of the epidermal growth factor receptor which elimithe endocytic pathway in normal cells. Flow sorting has been nates kinase activity causes the receptor and asignificant usedrecentlyto select CHO cell lineswithtemperatureportion of the ligand to be redirectedfrom the lysosomal conditional defects in the expression of membrane glycopropathway to the recycling pathway (23) whereas proteolytic teins on thecell surface (47). After screening several clonesfor a temperature-conditional processing of the insulin receptorat thecell surface results in redirection of the receptor from the recycling to the degradaloss of surface TfR, Tffl.11 was chosen for further analysis. tive pathway (48). Deletion of the extracellulargrowth factor This cell line shows temperature-dependent trapping of lahomology region of the low density lipoproteinreceptor inhibbeled Tf at 41 "C (Figs. 3 and 4) and a concomitant loss of its acid-dependent ligand dissociation and receptor recycling surface Tf binding (Fig. 5). Scatchard analysis shows that this loss of binding is causedbyadecrease in the number of (9). In addition, aggregation of receptors by multivalent ligands or antibodies causes a redirection of receptors to the surface receptors at the nonpermissive temperature (Table 111).Analysis of the clearanceof internalized Tf showed that lysosome (11,49). These datasuggest that a defect in the TfR TfI'l.ll has a temperature-sensitive defect in the rate of which causes aggregation or a change in conformation could receptor recycling (Fig. 6). After temperature shift, the initialresult in the loss of TfR from the surface. However, the fact rate of clearance of internalized Tf from TfTl.ll cells was that at 41 "C TfTl.11 shows a loss in surface a,M-receptor as well as TfR indicates that the defect in TfT1.ll is not reduced 5.5-fold compared with WTB cells, and 63% of the internal Tfwas released through a very slow component ( tl/z specific for TfR but is a more general defect in the pathway of receptor recycling. = 67 min; Table IV). It is not currently known if the Tf Although there are intrinsic properties of receptors which released through the slow process was intact or degraded. O'Keefe and Draper (44) described a mutant, AF192, iso- determine whether they will be recycled, once that decision lated frommouse L cells by selectingfor resistanceto a has been made there seems to be little distinction among diphtheria toxin-Tfconjugate, with an aberrant cycle. Tf Like receptors. The rates of externalization of several receptors, including Tf, a2M, and mannosylated proteins, are identical, suggesting that the receptors are moving as a single cohort 160 (50). Treatment withgrowth factors andphorbol esters causes a rapid redistribution of many receptors including TfR, aZM receptor, mannose 6-phosphate receptor, and mannose receptor from an intracellular pool to the plasma membrane (26, Orc 0 120 140 27,51), because of an increase in the rate constant for receptor externalization (26, 27). It is possible that TfTl.11 contains a defect in one of the components involved in regulating this rate. Isolation and further characterization of pleiotropic mutants defective in receptor recycling should lead to insights into the general mechanism of receptor recycling and permit \ identification of molecules required for this process. 1

-t

A-

Q

n

20

0

1

2

3

4 0

5

Time at 41 C (h) FIG. 7. T f f 1.11 cells lose surface a z M receptors at 41 "C. WTB (0)and TfTl.ll (A)cells were incubated a t 41 "C for the indicated times and labeled with Cy5-a2M at 41 "Cfor 6 min. Nonspecific uptake was less than 10% for all samples. Data shown are averages of duplicate samples from two separate experiments ( n = 4) normalized to WTB at33 "C.

Acknowledgments-We thank Greg LaRocca for technical assistanceandDrs. AlanWaggoner andLaurenErnst for the gift of Cy5.18OSu and advice on preparing Cy5 conjugates. We also thank Dr. Phil McCoy of the Pittsburgh Cancer Institute for assistance with the cell sorting. REFERENCES 1. Brown, M. S., Anderson, R. G. W., and Goldstein, J. L. (1983)

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