Li Z.,Thomas Jefferson University |
Jiao X.,Thomas Jefferson University |
Wang C.,Thomas Jefferson University |
Shirley L.A.,Thomas Jefferson University |
And 6 more authors.
Cancer Research | Year: 2010
The DNA damage response (DDR) activates downstream pathways including cell cycle checkpoints. The cyclin D1 gene is overexpressed or amplified in many human cancers and is required for gastrointestinal, breast, and skin tumors in murine models. A common polymorphism in the human cyclin D1 gene is alternatively spliced, resulting in cyclin D1a and D1b proteins that differ in their carboxyl terminus. Cyclin D1 overexpression enhances DNA damage-induced apoptosis. The role of cyclin D1 and the alternative splice form in regulating the DDR is not well understood. Herein cyclin D1a overexpression enhanced the DDR as characterized by induction of γH2AX phosphorylation, the assembly of DNA repair foci, specific recruitment of DNA repair factors to chromatin, and G2-M arrest. Cyclin D1 deletion in fibroblasts or small interfering RNA-mediated reduction of endogenous cyclin D1 in colon cancer cells reduced the 5-fluorouracil-mediated DDR. Mechanistic studies showed that cyclin D1a, like DNA repair factors, elicited the DDR when stably associated with chromatin. ©2010 AACR.
Makushok T.,University of California at San Francisco |
Alves P.,IGBMC |
Huisman S.M.,University of Zurich |
Kijowski A.R.,University of Zurich |
Brunner D.,University of Zurich
Cell | Year: 2016
Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-Actin but not the F-Actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization. © 2016 Elsevier Inc.
Nardini M.,University of Milan |
Gnesutta N.,University of Milan |
Donati G.,University of Milan |
Gatta R.,University of Milan |
And 8 more authors.
Cell | Year: 2013
The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways. © 2013 Elsevier Inc.
Sellier C.,IGBMC |
Sellier C.,College de France |
Freyermuth F.,IGBMC |
Tabet R.,IGBMC |
And 13 more authors.
Cell Reports | Year: 2013
Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery. Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the accumulation of mutant RNAs containing expanded CGG repeats. Charlet-Berguerand and colleagues now find that DROSHA-DGCR8, the enzymatic complex that processes microRNAs, is sequestered within nuclear aggregates of CGG RNA repeats. In addition, they show that the processing of microRNA is reduced in patients with FXTAS. These data suggest a model in which a human neurodegenerative disease is linked to sequestration of the microRNA-processing machinery. © 2013 The Authors.
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The following antibodies were used in this study: mouse-anti-actin (clone ac-15, Sigma-Aldrich, western blotting (WB): 1:10,000), mouse-anti-AP-1γ (γ-adaptin, clone 100/3, Sigma-Aldrich, immunofluorescence (IF): 1:100), mouse-anti-AP-2α (α-adaptin, clone AP-6, hybridoma cell line, IF: 1:100), rabbit-anti-APPL1 (Cell Signalling, IF: 1:100), mouse-anti-B10 (IGBMC, WB: 1:2,000), mouse-anti-β1-integrin (clone LM534, Millipore, IF: 1:375), mouse-anti-β1-tubulin (clone B5-1-2, Sigma-Aldrich, WB: 1:500), rabbit-anti-clathrin heavy chain (Abcam, IF: 1:500), mouse-anti-clathrin heavy chain (clone TD1, hybridoma cell line, WB: 1:500), rabbit-anti-EEA1 (Cell Signalling, IF: 1:100), mouse-anti-EGFR (clone R-1, Santa Cruz, IF: 1:100), mouse-anti-Exo70 (Millipore, WB: 1:500, IF: 1:100), rabbit-anti-Gadkin (ref. 25, WB: 1:1,000), mouse-anti-GM130 (BD Transduction, IF: 1:100), rabbit-anti-GFP (Abcam, WB: 1:10,000, IF: 1:500), mouse-anti-HA (clone HA.11, Covance, IF: 1:400), rabbit-anti-HA (Cayman Chemical, IF: 1:100), rabbit-anti-HA (clone Y-11, Santa Cruz, WB: 1:500), mouse-anti-HA-Alexa Fluor 488 (clone HA.11, Covance, IF: 1:100), mouse-anti-LAMP1 (BD Pharmingen, IF: 1:200), mouse-anti-LC3B (clone 4E12, MBL International, IF: 1:100), rabbit-anti-MTM1 (raised against amino acid 19-33 and amino acid 502-516 of human MTM1, WB: 1:250), mouse-anti-PI(4)P (catalogue: Z-P004, Echelon Biosciences, IF: 1:63), mouse-anti-PI(4,5)P (catalogue: Z-A045, Echelon Biosciences, IF: 1:200), mouse-anti-PI(3,4)P (catalogue: Z-P034b, Echelon Biosciences, IF: 1:150), rabbit-anti-PI4K2α (ref. 26, WB: 1:2,000), sheep-anti-PIKfyve (Tocris Biosciences, WB: 1:1,000), mouse-anti-Rab5 (BD Transduction, IF: 100), rabbit-anti-Rab7 (clone D95F2, Cell Signalling, IF: 1:50), rabbit-anti-Rab11a (Life Technologies, WB: 1:500), rabbit-anti-Sec3 (Proteintech Group, WB: 1:500), mouse-anti-Sec6 (Stressgen, WB: 1:500), mouse-anti-Sec8 (BD Transduction, WB: 1:500), mouse-anti-SNX4 (Sigma-Aldrich, WB: 1:500), mouse-anti-SNX17 (Proteintech Group, WB: 1:1,000), sheep-anti-TGN46 (Serotec, IF: 1:200), mouse-anti-TfR (clone H68.4, Life Technologies, IF/flow cytometry: 1:200), rabbit-anti-TfR (Sigma-Aldrich, IF: 1:100), rabbit-anti-Vps26 (Abcam, IF: 1:100), rabbit-anti-Vps34 (clone D9A5, Cell Signalling, WB: 1:1,000). All siRNAs used in this study were 21-, 23-, or 27-base oligonucleotides including 3′-dTdT overhangs. For silencing, the following siRNAs were used targeting the human isoform: Exo70 5′-GGTTAAAGGTGACTGATTA-3′, MTM1 5′-GATGCAAGACCCAGCGTAA-3′, MTMR1 5′-GAGATAGTGTGCAAGGATA-3′, MTMR2 5′-GGACATCGATTTCAACTAA-3′, MTMR4 5′-CAGCATAGGTTACGGCAAA-3′, MTMR7 5′-TGCAAGAACTTTCAGATAA-3′, PI4K2α 5′-GGATCATTGCTGTCTTCAA-3′, Rab11a 5′-AAGAGCGATATCGAGCTATAA-3′, Sec3 5′-CCTGTTGGATATGGGAAACAT-3′, Sec6 5′-CTGGAGGCAGAGCATCAACAC-3′, SNX4 5′-TGGTCAGAGTGTCCTAACA-3′, SNX17 5′-CTGGCTTTTGAATACCTCA-3′, and Vps34 5′-CCCATGAGATGTACTTGAACGTAAT-3′. For silencing Kif16b and PIKfyve, a pool of four siRNAs was obtained from Dharmacon (Thermo Scientific). The scrambled control siRNA used throughout this study corresponded to the scrambled γ1 adaptin sequence 5′-AAATCGGATATCGGAATAG-3′. Complementary DNA encoding full-length human MTM1 and MTMR2 was provided by G. Di Paolo and inserted into a pcDNA3.1(+)-based haemagglutinin (HA)-, mCherry-, or eGFP-expression vector with tags at the amino (N) terminus of MTM1 and MTMR2. siRNA-resistant MTM1 constructs were created by introducing four silent mutations: 5′-gatgc ag cc ag gtaa-3′. P205L, R241L, Y397C, and C375S mutants of MTM1 were created by mutation of the respective amino acid of human MTM1. N-terminally tagged human MTMR7 and mouse MTMR1 were in a pcDNA3.1(+)-based vector backbone. N-terminally tagged GFP–MTMR4 was a gift from M. Clague, N-terminally tagged GFP–Rab4A and GFP–Rab11A were a gift from P. van Sluis, and GFP–Rab14 was provided by T. Proikas-Cezanne. N-terminally tagged GFP–Rab5A GFP–Rab8A and GFP–Rab35 were in the pEGFP-C vector backbone (Clonetech) and Rab5A in a pcDNA3.1(+) vector backbone. Q79L mutant of Rab5A was created by mutation of the respective amino acid of human Rab5A. Full-length human SNX1, SNX3, SNX4, SNX8, SNX15, SNX17, and SNX27 were inserted in a pcDNA3.1(+)-based expression vector to express N-terminally tagged eGFP–fusion proteins except for carboxy (C)-terminally tagged SNX17–eGFP. N-terminally tagged eGFP–2xFYVE-Hrs, eGFP–2xPH-PLCδ and eGFP–2xPH-FAPP1 were in a pcDNA3.1(+)-based expression vector. The mRFP-2xPH-TAPP1 was a gift from T. Takenawa and the eGFP–2xPH domain of Bruton’s tyrosine kinase was a gift from M. Wymann. N-terminally tagged eGFP–mPI4K2α was in a pcDNA3.1(+)-based expression vector. N-terminally tagged HA-mPI4K2α was in the pcDNA5/FRT/TO expression vector (Life Technologies). D308A mutant of PI4K2α was created by mutation of the respective amino acid of mouse PI4K2α. To create N-terminally tagged GST-MTM1 and -Sec6, full-length human MTM1 and Sec6 were cloned in the Gateway pENTRA1 entry vector and by recombination transferred into the Gateway pGEX4T3 vector (Life Technologies). N-terminally tagged mouse PI4K2α was in the pGEX4T1 vector backbone. The B10-tag was engineered in-house by inserting the B10 epitope of the human oestrogen receptor in the pSG5 vector backbone (Stratagene) and used to create N-terminally tagged B10-MTM1 and B10-Sec6 (pSG5hERB10 empty vector). HeLa and COS-1 cells were from ATCC and not used beyond passage 30 from original derivation from ATCC without further authentication. Hek293 cells stably expressing HA-tagged mPI4K2α were generated using the FlpIn system developed by Life Technologies according to the manufacturer’s protocol. The fibroblast cell line H31 from patients with XLCNM has a genomic deletion of the entire MTM1 (refs 27, 28); the fibroblast cell line G92-628 from a patient with XLCNM, referred to as XLCNM patient 2, has a stop mutation in MTM1 at amino acid 37 (refs 27, 28). HDFa cells (human dermal fibroblast from adult healthy individuals) were obtained from Life Technologies. H31, G92-628, and HDFa cells were cultured in MEM (Life Technologies) containing 15% FCS and not used beyond passage 20. HeLa-M Clone 1 (C1) cells were cultured in DMEM (4.5 g l−1 glucose, Life Technologies) containing 10% FCS and 1.66 μg ml−1 puromycin and not used beyond passage 10 (ref. 29). All cell lines were routinely tested for mycoplasma contaminations on a monthly basis. HeLa cells were transfected with siRNA using Oligofectamin (Life Technologies) according to the manufacturer’s protocol. To achieve optimal knockdown efficiency, two rounds of silencing were performed. Cells were transfected on day 1, expanded on day 2, transfected for a second time on day 3, seeded for the experiment on day 4, and the experiment was performed on day 5. For transient overexpression of proteins in knockdown cells, plasmids were transfected on day 4 using Lipofectamin 2000 (Life Technologies) according to the manufacturer’s protocol. In case of co-transfection of two plasmids, JetPrime (Polyplus) was used according to the manufacturer’s protocol. For transient overexpression of proteins in untreated cells, plasmids were transfected 24 h before analysis using Lipofectamin 2000 (Life Technologies). For knockdown of transiently overexpressed proteins, cells were simultaneously transfected with plasmids and siRNA using Lipofectamin 2000 according to the manufacturer’s protocol; expression was allowed overnight and cells analysed the next day. HDFa, H31, and COS-1 cells were transfected using Lipofectamin 3000 (Life Technologies) according to the manufacturer’s protocol; expression was allowed overnight and cells analysed the next day. Cells seeded on coverslips coated with Matrigel (BD Biosciences) were serum-starved for 2 h and used for either Tf uptake or surface labelling. For quantitative Tf uptake, cells were treated with 25 μg ml−1 Tf-Alexa647 (Life Technologies) for 10 min at 37 °C. After being washed twice with ice-cold phosphate-buffered saline (PBS), cells were acid washed at pH 5.3 (0.2 M Na-acetate, 0.2 M NaCl) for 1 min on ice to remove surface-bound Tf, followed by washing twice with ice-cold PBS and fixation with 4% paraformaldehyde (PFA) for 45 min at room temperature (23–25 °C). For all immunocytochemistry stainings except MTM1 or Rab5 Q79L-expressing cells, inhibitor treatments (Wortmannin or Vps34-IN1), and staining of β1-integrin, cells were treated with 25 μg ml−1 Tf-Alexa647 (Life Technologies) for 30 min at 37 °C to allow Tf uptake to reach saturation. Cells were washed twice with PBS, followed by immunocytochemistry staining as described in Immunocytochemistry and spinning disc confocal imaging. For TfR surface labelling, cells were incubated with 25 μg ml−1 Tf-Alexa647 for 45 min at 4 °C to block endocytosis, washed three times with ice cold PBS, and fixed with 4% PFA for 45 min at room temperature. Tf uptake and surface labelling, and EGFR and LC3B labelling, were analysed using a Nikon Eclipse Ti microscope (eGFP filter set: F36-526; TexasRed filter set: F36-504; Cy5 filter set: F46-009; DAPI filter set: F46-000), equipped with a ×40 oil-immersion objective (Nikon), a sCMOS camera (Neo,Andor), and a 200 W mercury lamp (Lumen 200, Prior), operated by open-source ImageJ-based micromanager software and quantified using open-source ImageJ software. Levels of eGFR, LC3B, and internalized or surface-bound Tf were normalized to the cell area. The ratio of internalized Tf to surface-bound Tf was used to distinguish between uptake and recycling defects. Owing to small differences in TfR level in HDFa and H31 cells, surface-bound Tf was normalized to TfR level. TfR levels in HDFa and H31 cells were analysed by TfR antibody staining (see Immunocytochemistry and spinning disc confocal imaging) and analysed using a Nikon Eclipse Ti microscope as described above. Cultured cells seeded on Matrigel-coated coverslips were fixed for 10 min with 4% PFA, washed twice with PBS, permeabilized in blocking solution (10% goat serum, 20 mM Na H PO pH 7.4, 0.3% Triton X-100, 100 mM sodium chloride) for 30 min, and incubated with primary antibodies diluted in blocking solution for 1 h. After three washes with washing solution (20 mM Na H PO pH 7.4, 0.3% Triton X-100, 100 mM NaCl), secondary antibodies diluted in blocking solution were incubated for 1 h, followed by three washes in washing solution. For transient overexpression of eGFP–MTM1, cells were washed twice with ice-cold PBS, incubated with PEM (80 mM Pipes pH 6.8, 5 mM EGTA, 1 mM MgCl ) containing 0.05% saponin for 5 min at 4 °C, followed by a brief wash with ice-cold PEM. After fixation with 3% PFA for 15 min at 4 °C, cells were washed three times with PBS at room temperature, incubated with 50 mM NH Cl for 10 min and washed again twice with PBS. Immunocytochemistry was done as described above with the exception that Triton X-100 was exchanged for 0.05% saponin in all buffers. PI(3)P and PI(4)P stainings were performed as previously described30, although at a reduced concentration of eGFP–2xFYVE of 0.25 μg ml−1. If indicated, PI(3)P was labelled using a GST-tagged Phox domain of p40 chemically conjugated to Alexa Fluor 488, which was a gift from I. Ganley. For wortmannin (Sigma-Aldrich) treatment, cells were incubated with 2 μm wortmannin (dissolved in dry dimethylsulfoxide (DMSO)) or DMSO, diluted in serum-free medium, for 30 min at 37 °C, and subsequent PI(3)P staining was performed as described above. PI(3,4)P and PI(4,5)P stainings were performed as previously described31. For all LC3 immunocytochemistry stainings, fresh serum-containing medium was added 2 h before fixation. In case of bafilomycin A1 (BafA1, Sigma-Aldrich) treatment cells were washed three times with HBSS and incubated with 100 nM BafA1 (dissolved in dry DMSO) diluted in HBSS for 3 h. Control cells were incubated with DMSO diluted in serum-containing medium. After fixation with 4% PFA for 30 min, cells were washed three times with PBS, permeabilized with 200 μg ml−1 digitonin diluted in PBS for 10 min, followed by three washes with PBS. Cells were incubated with the primary antibody diluted in PBS for 1 h, followed by three washes with PBS. Secondary antibodies diluted in PBS were incubated for 1 h, followed by three washes in PBS. Protein and lipid immunocytochemistry stainings were routinely analysed and quantified using a spinning disc confocal microscope (Ultraview ERS, Perkin Elmer) with Volocity imaging software (Improvision, Perkin Elmer). For all quantifications, protein and lipid stainings were normalized to cell area. For quantification of TfR localization, cells with either perinuclear or peripheral TfR localization were counted. Peripheral TfR localization was defined as cells with either TfR dispersion or TfR accumulations at the cell periphery as shown in example images (Fig. 2c), and the normalized fraction of cells with perinuclear TfR localization was quantified. The amount of co-localization between two channels was quantified using thresholded Pearson’s correlation coefficients. To quantify the amount of co-localization at peripheral sites, thresholded Pearson’s coefficients were calculated in three randomly chosen 100 pixel × 100 pixel squares in the cell periphery. For averaged line scans, line profiles were calculated as the mean fluorescence intensity averaged over 100 pixels. Maximum intensity projections were calculated from z-stacks with 200 nm spacing between slices covering the whole cell. For statistical analysis, see Statistical analysis of immunocytochemistry and live-cell TIRF experiments. Cells seeded on Matrigel-coated coverslips were serum-starved for 30 min and treated with 1 μg ml−1 cholera toxin subunit B (Ctx) CF568 (Biotium) for 45 min at 37 °C, followed by 30 min chase in starvation medium. After being washed once with PBS, cells were fixed with 4% PFA for 30 min, washed twice with PBS, permeabilized in blocking solution (10% goat serum, 20 mM Na H PO pH 7.4, 0.3% Triton X-100, 100 mM sodium chloride) for 15 min and incubated with primary antibody diluted in blocking solution for 30 min. After three brief washes with PBS, secondary antibody diluted in blocking solution was incubated for 30 min, followed by three brief washes in PBS. C1 cells seeded on Matrigel-coated coverslips were washed once with PBS and treated with 1 μM D/D solubilizer (Clontech) diluted in HBSS to initiate secretion of the reporter construct. To halt secretion at the indicated time points, cells were placed on ice, washed twice with ice-cold PBS containing 10 mM MgCl and fixed with 4% PFA for 20 min at room temperature, followed by washing twice with PBS. Secretion of the GFP-tagged reporter construct was analysed using a Nikon Eclipse Ti microscope (see Transferrin uptake and surface labelling). Fifteen minutes after initiating secretion the reporter is localized at the Golgi complex. To calculate the secretion from the Golgi, all time points were normalized to 15 min. TIRF microscopy was performed using a Nikon Eclipse Ti microscope, equipped with an incubation chamber (37 °C), a ×60 TIRF objective (oil-immersion, Nikon), a sCMOS camera (Neo, Andor), a 200 W mercury lamp (Lumen 200, Prior), a triple-colour TIRF setup (laser lines: 488 nm, 568 nm, 647 nm), and operated by open-source ImageJ-based micromanager software. Cells seeded on Matrigel-coated coverslips were treated with 50 μg ml−1 Tf-Alexa647 (Life Technologies), diluted in serum-free medium for 30 min at 37 °C and 5% CO . For analysis of transferrin exocytosis, time-lapse movies of 15–30 s with a frame rate of 5 Hz were recorded. For all time-lapse movies, the 488 nm channel was acquired before the 647 nm channel, except for Extended Data Fig. 9a, g. Fusion events with the plasma membrane were defined by their characteristic time course (appearance, broadening/spreading of the fluorescence signal, and disappearance), counted and normalized to cell area. Representative kymographs were chosen over 30 s along a line of 400 pixels in length. For statistical analysis, see Statistical analysis of immunocytochemistry and live-cell TIRF experiments. Cell-permeable acetoxy methylester (AM)-protected phosphatidylinositol derivatives were synthesized according to published procedures17. For treatment of cells, PI(3)P/AM and PI(4)P/AM were dissolved in dry DMSO and mixed with an equal volume of 10% pluronic F127 in DMSO (Sigma-Aldrich). PIP/AMs were diluted in serum-free medium to a final concentration of 100 μM. For immunocytochemistry stainings, cells seeded on Matrigel-coated coverslips were treated with DMSO + pluronic F127 (control) or PIP/AMs for 30 min at 37 °C and then processed as described above. For TfR immunocytochemistry stainings, cells were stimulated with 25 μg ml−1 Tf-Alexa647 during the PIP/AM treatment. For live-cell TIRF imaging, cells seeded on Matrigel-coated coverslips were treated with 25 μg ml−1 Tf-Alexa647 and DMSO + pluronic F127 (control) or PIP/AMs for 30 min at 37 °C, directly followed by live-cell imaging. For the analysis of β1-integrin accumulations, H31 or HDFa cells were seeded on Matrigel-coated coverslips and treated with DMSO or Vps34-IN1 at a concentration of 0.01–1 μM dissolved in dry DMSO and diluted in serum-containing medium for 48 h, adding freshly diluted Vps34-IN1 or DMSO after 24 h. β1-integrin staining was performed as described in Immunocytochemistry and spinning disc confocal imaging. For live-cell TIRF imaging (see Fig. 3d), cells seeded on Matrigel-coated coverslips were treated with 50 μg ml−1 Tf-Alexa647 and DMSO or 1 μM VPS34-IN1 for 60 min at 37 °C, directly followed by live-cell imaging in the presence of DMSO or VPS34-IN1, respectively. Human EGF was purchased from Peprotech and 125I-labelled EGF from Perkin Elmer. 125I-labelled EGF degradation was performed as described previously32. Human holo-transferrin was purchased from Sigma-Aldrich and 125I-labelled Tf from Perkin Elmer. HeLa cells seeded in 24-well plates were starved for 1 h in serum-free medium containing 0.1% BSA and 20 mM HEPES pH 7.4. Cells were stimulated with 1 μg ml−1 125I-labelled Tf in starvation medium at 37 °C for the indicated time points and washed twice on ice with PBS. Surface-bound 125I-labelled Tf was removed by an acid wash with 0.2 M acetic acid, 0.5 M NaCl for 5 min on ice, collected, and radioactivity was measured using a scintillation counter (HIDEX 300SL). Cells were dried at room temperature for 5 min, lysed with 1 M NaOH for 60 min and the radioactivity of the lysate measured (corresponding to internalized Tf). Non-specific binding was measured for each time-point in the presence of a 300-fold excess of cold Tf and was subtracted from all values. The ratio of internalized to surface-bound Tf was plotted over time and Michaelis constant (K ) values were calculated using Prism software (GraphPad). On ice, cells were washed once with ice-cold PBS and detached from the culture dish by incubating for 5 min on ice with 0.1% PronaseE (Sigma-Aldrich), 0.5 mM EDTA solution in PBS. Cells were resuspended in PBS, pelleted at 300 g for 5 min at 4 °C and fixed in 4% PFA, 4% sucrose in PBS for 20 min at room temperature. Ten times excess volume of blocking solution (0.05% Saponin, 0.01% BSA in PBS) was added, then cells were pelleted and resuspended in blocking solution. After 15 min primary antibody (mouse-anti-TfR) was added and incubated for an additional 1 h. After washing once with blocking solution, cells were incubated for 1 h with secondary antibody diluted in blocking solution, followed by washing once with 0.2% BSA in PBS. Cells were resuspended in 0.2% BSA in PBS and analysed by flow cytometry using a BD LSRFortessa. Cells from a 100% confluent 10 cm cell culture dish were harvested in homogenization buffer (20 mM HEPES pH 7.4, 100 mM KCl, 2 mM MgCl , 1 mM PMSF, 0.1% protease inhibitor cocktail) and homogenized using a European Molecular Biology Laboratory cell cracker (HGM; inner diameter 8.020 mm, ball diameter 8.004 mm, with 12 strokes), followed by three freeze–thaw cycles in liquid nitrogen. Total cell lysate was collected after centrifugation at 1,000 g for 5 min at 4 °C. To obtain the cytosolic fraction, total cell lysate was centrifuged at 100,000 g for 30 min at 4 °C, the supernatant was collected, and protein concentration and volume determined. The membrane pellet was washed once in homogenization buffer and collected in a volume corresponding to the volume of the cytosol fraction. Equal volumes of total cell lysate (minimal concentration 0.125 μg μl−1, corresponding to minimal loading of 5 μg), membrane pellet, and cytosol fraction were loaded onto a 10% acrylamide gel for SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblotting. Western blot development was done using a LI-COR Odyssey Fc imager, and western blot bands were quantified using Image Studio Lite Version 4.0 software (LI-COR). For Exo70, Sec3, Sec8, and MTM1, protein levels in the total cell lysate were normalized to actin, whereas protein levels in the membrane fraction were normalized to gadkin. The ratio of membrane to total cell lysate was used to quantify the membrane fraction of Exo70, Sec3, Sec8, and MTM1. All knockdown conditions were normalized to membrane fractions of the respective protein in scrambled siRNA-treated controls. Data are presented as mean values ± s.e.m. from five independent experiments (n). Statistical testing was performed using a one-sample t-test. Expression of HA-tagged PI4K2α in stably transfected Hek FlpIn cells was induced overnight by addition of doxycylin. As a control, untransfected Hek FlpIn cells were used. Cells were harvested in lysis buffer (20 mM HEPES pH 7.4, 100 mM KCl, 2 mM MgCl , 1 mM PMSF, 0.1% protease inhibitor cocktail, 1% Triton X-100) and incubated on ice for 30 min, followed by centrifugation at 43,500 g for 20 min at 4 °C. The supernatant was centrifuged again at 265,000 g for 15 min at 4 °C. HA-matrix beads (Covance, mouse-anti-HA.11) were used to immunoprecipitate HA-tagged PI4K2α. Protein (3–5 mg ml−1) was loaded on 30 μl 1:1 washed HA-matrix beads slurry and incubated for 1 h at 4 °C on a rotating wheel. Beads were pelleted, washed twice with lysis buffer, followed by two washes with homogenization buffer, and bound protein was eluted in 60 μl 1× Laemmli sample buffer. Eluates were loaded onto a 10% acrylamide gel for SDS–PAGE followed by immunoblotting. Pulldown assays using GST-tagged PI4K2α as a bait were performed as described previously26. For pulldown assays using GST-tagged MTM1 and Sec6, GST fusion proteins were expressed in Escherichia coli BL21 pRARE strain. For negative control, the empty pGEX4T3 vector (GST alone) was used. The induction of expression was performed with 1 mM IPTG at 16 °C for 14 h. Bacteria were lysed on ice by sonication in lysis buffer (50 mM Tris HCl pH7, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, complete protease inhibitor cocktail (PIC, Roche)) supplemented with 1 mg ml−1 lysozyme, 1 mM PMSF, 0.1% sarcosyl, 0.5% Triton X-100, and then centrifuged at 15,000 g for 30 min. GST-tagged proteins were purified from bacterial lysates by incubation with Glutathione Sepharose 4B beads (GE Healthcare) for 1 h followed by extensive washing with lysis buffer plus 1 mM PMSF. In parallel, COS-1 cells expressing B10-MTM1, B10-SEC6, HA-PI4K2α, or corresponding controls were lysed with a buffer containing 20 mM HEPES-KOH pH 7.5, 200 mM NaCl, 0.5% Triton X-100, 10% glycerol, 4 mM DTT, 1 mM EDTA, and PIC by passage through 25-gauge needles. Insoluble material was removed by centrifugation at 11,000 g for 10 min. Twenty micrograms of the purified GST fusion proteins coupled to glutathione beads were then incubated with 350 μg of COS-1 cell extracts. After washing beads three times with a buffer containing 20 mM HEPES-KOH, 200 mM NaCl, 1 mM DTT, and PIC (pH 7.3), 10 μg of GST beads were analysed by electrophoresis on a 10% SDS–polyacrylamide gel. Bound B10-tagged MTM1 and Sec6 or HA-tagged PI4K2α were detected with a B10-specific antibody or a PI4K2α-specific antibody, respectively. The entire procedure was performed at 4 °C, unless specified. Liposome flotation assays were performed in Hek293 cells as previously described31. Scrambled or MTM1 siRNA transfection was performed as described in HeLa cells that were grown in 6 cm plastic dishes. Cells were serum-starved for 2 h and treated with 25 μg ml−1 transferrin-HRP (Fitzgerald) for 30 min at 37 °C. Cells were fixed with 2% glutaraldehyde in PBS. After rinsing in fresh PBS, cells were mechanically detached by scratching, pelleted, and embedded into gelatine. After osmification with aqueous 1% osmium tetroxide, samples were stained en bloc with 1% aqueous uranil acetate and embedded in epoxy resin. Sections were viewed with Zeiss 910 transmission electron microscope and micrographs were taken along the cell perimeter at ×20,000. For morphometric analysis, images were combined to reconstruct the perimeter of a cell. The size and number of transferrin-HRP-labelled organelles up to 1 μm distance from the plasma membrane were quantified. The number of Tf-HRP endosomes was normalized to the plasma membrane perimeter. Ten cells per condition were analysed. To calculate the size of Tf-HRP endosomes, 52 endosomes for scrambled siRNA-treated controls and 90 endosomes for MTM1-depleted cells from a total of 10 cells were analysed. Data are presented as mean values ± s.e.m. A statistical test was performed using a one-sample t-test. For analysis of immunocytochemistry experiments, a minimum of three independent experiments (n) was performed and statistically significant estimates for each sample were obtained by choosing an appropriate sample size, correlating to 15–30 images per condition per experiment for microscopy-based quantifications. Cells were chosen arbitrarily according to the fluorescent signal in a separate channel, which was not used for quantification. Data are presented as mean values ± s.e.m. For analysis of exocytic events per unit area in live-cell TIRF imaging experiments, a minimum of ten videos (duplicate coverslips: five videos per coverslip) per condition per experiment were acquired and fusion events were counted in minimally five videos per condition per experiment. A minimum of three independent experiments were performed and data represent mean values ± s.e.m. All statistical tests were performed using a two-tailed, unpaired t-test, without excluding samples from statistical analysis.