Controlled Elimination of Clathrin Heavy-Chain Expression in DT40 Lymphocytes Frank R. Wettey, Steve F. C. Hawkins, Abigail Stewart, J. Paul Luzio, Jonathan C. Howard, and Antony P. Jackson

Cell culture and molecular biology protocols. Except where indicated, DT40 cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 2 gl^-1 NaHCO₃, 10% heat-inactivated fetal calf serum and 1% heat-inactivated chicken serum. Cells were grown at 37^°C in a 5% CO₂ atmosphere. Chicken brain cDNA and genomic libraries (Clontech) were screened with the rat clathrin heavy-chain cDNA labeled with ³²P (S1) and sequenced using the automated facilities of the Department of Biochemistry, Cambridge. Southern blots were performed as described (S1). Vectors containing neomycin and hygromycin resistance genes (designated pSNC and pSHC) were constructed by inserting the resistance genes into a BamHI site of a 5.3 kbp SacI fragment of chicken genomic clathrin DNA (Fig. S1A, S1B). DT40 cells were transfected by electroporation, selected with antibiotic and transfectants sub-cloned as described (S2).

Immune blotting. Cells were lysed in 30 mM Tris HCl, pH 7.6, 75 mM NaCl, 1% Triton X-100, 0.5 % sodium deoxycholate, 0.1% SDS, 0.1 mg ml^-1 PMSF, 1 mM sodium ortho-vanadate, 50 mM sodium fluoride, 10mM sodium--glycerophosphate, 5mM sodium pyrophosphate, 1:200 Protease inhibitor Cocktail (Sigma) and centrifuged briefly to remove cell debris and nuclei. About 60 g of protein extract was separated by SDS PAGE in 10% polyacrylamide gels and transferred onto nitrocellulose membranes (S1). Western blots were visualised by enhanced chemiluminescence (Pierce SuperSignal Kit) and where necessary, quantified with a Leica Q500 image analyser and software.

Apoptosis assays. Apoptotic body staining: About 2.5x10⁵ cells were centrifuged, washed in phosphate-buffered saline (PBS) and centrifuged onto poly-lysine-coated glass slides. Cells were fixed in PBS containing 3.7% paraformaldehyde for 10 minutes, stained with 2.5 g ml^-1 Hoechst 33342 and examined by fluorescence microscopy. DNA laddering: Genomic DNA was extracted from 3x10⁶ cells with lysis buffer (100 mM Tris HCL pH 8.5, 5 mM EDTA, 0.2%SDS, 200 mM NaCl, 100 g ml^-1proteinase K) (S1). Approximately 7 g DNA samples were separated by electrophoresis on 1.5% agarose, stained with ethidium bromide and photographed under UV illumination. Caspase activity: Cells (7.5x10⁵) were centrifuged (3 min, 300g) and lysed in 150l hypotonic buffer (10 mM Hepes, pH 7.4, 42 mM KCl, 5 mM MgCl₂, 1 mM DTT, 1 µg ml^-1 pepstatin A, 1 µg ml^-1 leupeptin, 5 µg ml^-1 aprotinin, and 0.1% Triton X-100). 10l per sample were assayed with 150l of caspase assay buffer (20 mM Hepes, pH 7.4, 1 mM EDTA, 10% sucrose, 5 mM DTT, 0.1% Chaps, and 13.3 µM DEVD.AMC (Research Biochemicals International). After 5 hours, cleavage of DEVC.AMC was determined with a Packard fluorescent plate reader (360nm_ex/460nm_em) (S3).

Receptor-mediated endocytosis. Iron-saturated conalbumin was labeled with ¹²⁵I using Chloramine-T (S4). Cells were washed twice in PBS, incubated for 20 minutes at 37°C in Buffer A (RPMI 1640 medium without phenol red,10mM HEPES, 0.1% BSA, pH 7.4), washed twice in ice-cold Buffer A and resuspended in ice-cold Incubation Buffer (RPMI 1640 medium without phenol red, 20mM HEPES, 5mM glucose, 0.1% BSA, pH 7.4). Cells (1 x 10⁷ ml^-1) were mixed with ¹²⁵I-conalbumin (4 x 10⁷ cpm ml^-1 20 nM) at 4°C. After 1 hour, the cells were shifted in 100µl aliquots to 37°C and the uptake stopped at the indicated times by the addition of 900 µl ice-cold PBS/0.1% BSA and transfer to iced water. The cells were washed a further five times in PBS, solubilised in 1ml PBS/0.1% Triton X-100 and the radioactivity quantified in a scintillation counter. The relative accumulation of label was calculated from : (total counts in pellet at time t – counts in pellet at time 0)/ counts in pellet at time 0. A hundred-fold excess of unlabelled conalbumin completely inhibited ¹²⁵I-conalbumin uptake.

Fluid-phase endocytosis. Cells (2x10⁶ cells ml^-1) were washed twice in ice-cold Buffer A containing 0.2% BSA and resuspended at in Buffer A containing 0.2% BSA and 1mg ml^-1 HRP type VI (Sigma). At the indicated times 0.5ml aliquots of the suspension were transferred from 37^°C to 1ml of ice-cold Wash Buffer (PBS, 0.1% BSA,1mM EDTA), centrifuged and washed three times. Surface-attached HRP was removed with 10mM Tris HCl, 150mM NaCl, 5mM KCl, 1mM EDTA, pH 7.4 containing 1 mg/ml Proteinase K (Roche) for 1hr at 4°C followed by 20 seconds at 37^°C. The cells were washed three times in PBS containing 10 g ml^-1 PMSF. Pellets were solubilised in PBS, 0.2% Triton X-100, assayed for internalised enzyme using o-phenylenediamine substrate and quantified at 450nm. Amount of HRP uptake in pg was calculated by comparison to a curve generated from serially diluted HRP standard. Endogenous peroxidase activity was determined in the absence of HRP and subtracted from all values.

Conalbumin recycling. Cells depleted of endogenous transferrin (see above) were incubated for 20 minutes at 37^°C with ¹²⁵I-labelled conalbumin (4 x 10⁷ cpm ml^-1), chilled for 15 minutes on ice and washed three times in ice-cold PBS containing 0.1 % BSA. Surface label was stripped by an acid wash (50 mM glycine, 150mM NaCl, 0.9 mM CaCl₂, 0.5 mM MgCl₂, pH 3.0), neutralised with 5 volumes of RPMI 1640 medium without phenol red, 20 mM HEPES, 10% Chicken Serum pH 7.4, spun and washed twice as above and resuspended in Incubation Buffer at 10⁷ cells ml⁷ containing 100-fold excess of unlabelled conalbumin. Cells were shifted in 0.1 ml aliquots to 37^°C and then stopped at the indicated times by the addition of 0.9 ml ice-cold PBS/0.1% BSA. After centrifugation, pellets were lysed in PBS containing 0.1% Triton X-100. The radioactivity in pellets and supernatants were measured by scintillation counting. For each point, the combined cell-associated and medium-derived counts were constant.

Immunofluorescence. Cells were spun (175 x g, 1 minute) in chambered poly-l lysine-coated slides to adhere cells to the slide surface and prepared for immunofluorescence as described previously (S5), except that 2.5 g/ml Hoechst 33342 was included in the last wash. Equivalent optical sections were scanned and analysed with a Leica TCS-NT confocal microscope.

-glucuronidase assay. Cells (1.5x10⁶ cells ml^-1) were washed twice in PBS and resuspended in Buffer B (RPMI 1640 medium without phenol red, 0.1% BSA, containing 10 mM mannose 6-phosphate pH 7.4). After incubation of 0.5ml aliquots at 37°C for 0 and 6 hrs, 0.5ml of ice-cold Buffer B was added, the cells spun at 400 g, supernatant retained, and the cell pellet lysed in 100mM Na-citrate buffer, 0.1% Triton X-100 pH 5.5. Cell lysates were incubated with 4-methylumbelliferyl--D-glucuronide for 100 minutes and the release of 4-methylumbelliferone from the supernatant and lysate fractions determined fluorimetrically with a Perkin-Elmer MPF-44F spectrophotometer at 390nm_ex/480nm_em. Missorting was calculated as the difference between time 0 and 6 hrs and expressed as -glucuronidase activity measured in the supernatant as a percentage of the total.

Organelle separation on Percoll gradients. Cells were washed twice in PBS and twice in HB Buffer (250mM Sucrose, 20mM HEPES, 1mM EDTA, 1mM Mg Acetate, pH 7.5). After resuspending the cell pellet in 4 volumes of ice-cold HB Buffer containing 1:100 Protease Inhibitor Cocktail (Sigma), the cells were broken by 4-8 strokes through a 27G-needle to achieve approximately 60% breakage. DNaseI was added at 1mg/ml and incubated 10min at 25°C prior to spinning out the nuclei twice for 10min at 2000g, 4°C. About 3ml of supernatant were layered onto 10ml 20%Percoll buffered in HB buffer in 13.5ml Beckman quick-seal centrifuge tubes (S6). Gradients were spun for 20min at 19.500 rpm at 4°C in a Beckman 50Ti rotor. Fractions were collected from the bottom and stored at -80°C. Activity of -glucoronidase was determined as above, galactosyl-transferase and lactate-hydrogenase activity were assayed as described in (S7).

BSA gold-labelling and immunoelectron microscopy. BSA conjugated to 5 nm colloidal gold was prepared as described previously (S8). Cells were incubated with the conjugate-containing medium for 4 hours at 37^°C followed by incubation in conjugate-free medium for 20 hours and 70 nm sections observed in a Philips CM100 electron microscope. The mean area of the lysosomes was measured using the online facility of the Philips CM100 microscope at a magnification of 4600x. Between 75 and 100 BSA-gold-positive structures were scored for each sample. For immunogold electron microscopy, cells were fixed with 8% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2 at room temperature for 1 hour. Ultrathin frozen sections (50 nm) were collected from the knife-edge with 50:50 2% methyl cellulose: 2.3 M sucrose, immunolabeled, contrasted, dried and observed as above.

Supplemental Figure 1. A: Partial restriction map of the chicken clathrin heavy-chain (Chc) gene showing the location of the gene targeting cassettes (pSNC and pSHC) used. In both cassettes, the antibiotic resistance gene was flanked by a 1.5 kb SacI-BamHI and a 3.5 kb BamHI genomic insert. B: Flow diagram of the experimental strategy used to sequentially construct the DKO-S cell line. C: Southern blot of SacI-digested genomic DNA isolated from wild type DT40 (+/+), a heterozygous neomycin-resistant intermediate (+/-) and DKO-S cells (-/-). Southern blot was probed under high stringency conditions using ³²P-labelled 0.4 bp BamHI-SacI fragment as shown in A.

Figure 1A

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Figure 1B

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Figure 1C

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Supplemental Figure 2. Additional evidence for apoptosis in clathrin-depleted DKO-S cells. A: DKO-S cells were incubated with or without 50 ng ml^-1 doxycycline (dox) for 72 hours, fixed and stained with 2.5 g ml^-1 Hoechst 33342 and examined by fluorescence microscopy. Arrows indicate apoptotic bodies B: DNA degradation in DKO-S cells. Genomic DNA was isolated from DKO-S cells at the indicated times after treatment either with or without 50 ng ml^-1 doxycycline and analysed as described in supplementary materials and methods. Note that the caspase inhibitor BAF delays appearance of DNA fragmentation in clathrin-depleted cells.

Figure 2A

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Figure 2B

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Supplemental Figure 3. The 68 kDa band intensity reflects Akt pathway activation. Separate aliquots of DKO-S cells (10⁵ml^-1) were either mock-treated, incubated with 100 M LY294002 (a class I PI3 kinase inhibitor) for 20 miunutes at 37^°C prior to the addition of cross-linking anti-chicken IgM monoclonal antibody M1 at 6 g ml^-1 for 30 minutes or with M1 antibody only. Cross-linking surface IgM on DT40 cells is known to activate the Akt pathway (S9).

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Supplemental Figure 4. Confocal immunofluorescence microscopy analysis of LEP100 distribution in DKO-R cells grown without (A) or with (B) 50ng ml^-1 doxycycline for 96 hours. Nuclei were stained with 2.5 g/ml Hoechst 33342. Bar = 5m.

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