Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: VE-cadherin endocytosis is mediated through a clathrin-, AP-2–, and dynamin-dependent pathway. (A and B) Cell surface IL-2R-VE-cadcyto was labeled with IL-2R antibodies at 4°C. Cells were incubated at 37°C for 5 min, washed in a low pH buffer at 4°C to remove surface bound antibody, and then processed for immunofluorescence to visualize intracellular IL-2R-VE-cadcyto (A, a′, c′, and e′). Antibodies against the myc epitope tag at the carboxyl terminal domain of the IL-2R-VE-cadcyto were used to verify expression of the polypeptide (A, b′, d′, and f′). Note that untreated cells exhibited high levels of internalized IL-2R-VE-cadcyto, whereas treatment with K+ depletion or chlorpromazine completely inhibited internalization. (C–F) MECs were infected with adenoviruses expressing either GFP (C, a′ and b′) or dominant negative DynII (C, c′ and d′). The DynII completely blocked internalization of Alexa Fluor 555–conjugated transferrin (C and D). To monitor internalization of VE-cadherin, MECs were either untreated (E, a′ and b′) or treated with 100 μM chloroquine for 3 h (E, c′–f′). Note that intracellular vesicular accumulation of VE-cadherin is reduced in cells expressing DynII (E, e′ and f′, and F). (G–K) HMEC-1 cells were transfected with an siRNA oligo targeted to the AP-2 μ subunit or a nontargeting control siRNA. Lysates prepared from cells treated with control siRNA or AP-2 siRNA were separated by SDS-PAGE and immunoblotted for AP-2 to confirm AP-2 knockdown (G). Western blot and immunofluorescence reveals ∼75% reduction in AP-2 levels in siRNA-treated cells compared with control, as well as an 85% decrease in transferrin internalization (G–I). IL-2R-VE-cadcyto internalization assays were performed and AP-2 siRNA-treated cells (J, c′ and d′) exhibited an 80% decrease in internalization of IL-2R-VE-cadcyto compared with control (J, a′ and b′), indicating a requirement for AP-2 in VE-cadherin internalization (K). Error bars, SEM; n > 20 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Labeling, Incubation, Immunofluorescence, Expressing, Infection, Dominant Negative Mutation, Transfection, SDS Page, Western Blot

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: The VE-cadherin cytoplasmic tail specifically associates with clathrin and AP-2. MECs expressing IL-2R or IL-2R-VE-cadcyto were treated with DSP (lanes 2, 4, 7–8, and 11–12) or DMSO vehicle (lanes 1, 3, 5–6, 9–10, and 13–14), and extracted in detergent, and proteins were immunoprecipitated with beads alone (lanes 5–8), beads coated with AP-2 α antibodies (lanes 8–12), or beads coated with clathrin antibodies (CHC; lanes 13–14). Western blot analysis using antibodies against IL-2R to detect IL-2R or IL-2R-VE-cadcyto demonstrates that the IL2R-VE-cadcyto specifically interacts with AP-2 (lane 12) and clathrin (lane 14). Inputs represent 5% of sample.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: The VE-cadherin cytoplasmic tail specifically colocalizes with clathrin and AP-2. MECs expressing IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto were labeled for 30 min at 4°C with IL-2R antibodies, transferred to 37°C for 5 min, and then processed for immunofluorescence microscopy. As a positive control, internalization of transferrin receptor was monitored by labeling MECs with fluorescently conjugated transferrin at 4°C, transferring cells to 37°C for 5 min, and then processing for immunofluorescence microscopy. (A and B) Colocalization of transferrin, IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto with clathrin or caveolin was monitored. Colocalization was quantified as the percentage of transferrin, IL-2R, IL-2R-VE-cadcyto, or IL-2R-Dsg3cyto that colocalize with clathrin or caveolin, using Metamorph software. (C and D) Colocalization of IL-2R and IL-2R-VE-cadcyto with AP-2 was also measured. Colocalization was quantified as the percentage of transferrin, IL-2R, or IL-2R-VE-cadcyto that colocalize with AP-2. Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Labeling, Immunofluorescence, Microscopy, Positive Control, Transferring, Software

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: Exogenous expression of p120 prevents IL-2R-VE-cadcyto from colocalizing with clathrin and AP-2 in a manner dependent on the interaction of p120 with the VE-cadherin JMD. (A) p120 was coexpressed in MECs with either IL-2R-VE-cadcyto or IL-2R-VE-cadJMD-AAA, which is unable to bind to p120, and internalization assays were conducted as described in Figure 3. Cells were fixed and processed for triple-label immunofluorescence to monitor the IL-2R-VE-cadherin polypeptides, p120, and clathrin. (B) Colocalization was quantified as the percentage of IL-2R-VE-cadcyto or IL-2R-VE-cadJMD-AAA that colocalize with clathrin. In the presence of p120, colocalization between IL-2R-VE-cadcyto and clathrin is greatly reduced. High levels of colocalization between IL-2R-VE-cadJMD-AAA and clathrin in cells expressing exogenous p120 demonstrate that p120 must bind to the VE-cadherin JMD to prevent VE-cadherin recruitment into clathrin-enriched membrane domains. (C) Colocalization between IL-2R-VE-cadcyto and AP-2 was also monitored. In the presence of p120, IL-2R-VE-cadcyto colocalization with AP-2 is dramatically reduced (D). Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Expressing, Immunofluorescence

Journal:

Article Title: p120-Catenin Inhibits VE-Cadherin Internalization through a Rho-independent Mechanism

doi: 10.1091/mbc.E08-07-0735

Figure Lengend Snippet: A p120 Rho-uncoupled mutant prevents entry of VE-cadherin into membrane compartments containing clathrin and AP-2. (A and C) MECs were infected with adenovirus carrying IL-2R-VE-cadcyto and either p120 1A or p120 4A K622,628A. Cells were then processed for immunofluorescence to determine the extent of colocalization between IL-2R-VE-cadcyto and clathrin (A) or AP-2 (C) in cells expressing either wild-type or Rho-uncoupled p120 (p120 4A K622,628A). Colocalization was quantified as the percentage of IL-2R-VE-cadcyto that colocalize with clathrin or AP-2. The RhoA-uncoupled p120 mutant was as effective at preventing colocalization of IL-2R-VE-cadcyto as wild-type p120 (B and D). Error bars, SEM; n = 25 cells. Scale bar, 20 μm.

Article Snippet: The supernatants were incubated overnight at 4°C with Dynal magnetic beads (Invitrogen) conjugated to mAbs against AP-2 (α subunit, BD Transduction Laboratories) or clathrin (Calbiochem, San Diego, CA).

Techniques: Mutagenesis, Infection, Immunofluorescence, Expressing

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A ) Whole cell lysates from wild-type (WT) HeLa SS6 cells and TALEN clones #6, #27, #52, #64, #68 and #71 were analyzed by SDS-PAGE. Duplicate immunoblots were probed with polyclonal antibodies directed against FCHO2, the AP-2 μ2 subunit and epsin 1. ( B and C ) Selected but representative confocal optical sections of HeLa SS6 cells either mock transfected ( B ) or transfected with FCHO2 transcript-targeting siRNA oligonucleotides ( C ). Fixed cells were stained with a mAb directed against the AP-2 α subunit (AP.6, green) and affinity purified antibodies against DAB2 (red). ( D – K ) HeLa SS6 cells ( D ) or the indicated TALEN-treated clones ( E – K ) were fixed and stained with mAb AP.6 (green) and affinity purified antibodies directed EPS15 (red). Color-separated channels from a portion of the micrograph of clone #64 cells ( H ) are presented ( I ). Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.04137.004

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Clone Assay, SDS Page, Western Blot, Transfection, Staining, Affinity Purification

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A ) Semi-quantitiative RT-PCT analysis of muniscin protein transcripts in parental HeLa SS6, clone #64 and clone#64/1.E cells. The same PCR primers as in were used. HC; heavy chain. ( B ) Whole cell lysates from HeLa SS6, clone #64 and K562 cells were resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with antibodies specific for the indicated proteins. Positions of the molecular mass standards (in kDa) are indicated on the left. ( C and D ). After incubation at 37°C for 60 min in serum-free medium, cover slip-attached HeLa SS6 ( C ) or clone #64 cells ( D ) were pulsed for 10 min with Alexa Fluor488-labelled transferrin (green). After chilling on ice and washing with ice-cold PBS, the cells were fixed and stained for AP-2 (red) with the α-subunit specific mAb AP.6. Representative single confocal optical sections focused on a medial region, rich in transferrin-positive endosomal structures, are shown, with color-separated channels of the boxed regions shown below. Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.04137.005

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: SDS Page, Staining, Incubation

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A – C ) HeLa SS6 cells were either mock transfected ( A ) or transfected with siRNA oligonucleotides directed against the FCHO1 ( B ) or FCHO2 ( C ) mRNA . Silenced cells were fixed and stained with an antibody (mAb AP.6) against the α subunit of the AP-2 adaptor complex. Note that the randomly-scattered similarly-sized clathrin-coated structures typical of control HeLa SS6 cells are also present in FCHO1-silenced cells. The FCHO2-depleted cells exhibit the characteristic enlarged and more sparsely arrayed clathrin patches, analogous to FCHO2 gene-edited HeLa cone #64 cells. The efficacy of FCHO1 and FCHO2 transcript silencing using this siRNA protocol has been documented . DOI: http://dx.doi.org/10.7554/eLife.04137.007

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Transfection, Staining

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A and B ) Representative confocal optical sections of HeLa SS6 ( A ) or clone #64/1.E ( B ) cells incubated with 25 μg/ml Alexa Fluor488-conjugated transferrin (green) for 2 min at 37°C before washing on ice. Fixed cells were stained with the anti-AP-2 α subunit mAb AP.6 (red). The borders of some cells in the field are outlined (orange) and color-separated views of the boxed regions are shown on the right. Scale bar (for A – D ): 10 μm. ( C and D ) Analogous images from control HeLa SS6 ( C ) or clone #64/1.E cells ( D ) after a 10-min pulse of transferrin. ( E – H ) Confocal image of adherent ventral region of HeLa clone #64/1.E cells transiently transfected with GFP-FCHO1 (1-889) (green) before addition of a 2-min pulse of 25 μg/ml Alexa Fluor568-labeled transferrin (red) as in A and B . Fixed cells were immunolabeled for AP-2 with mAb AP.6 (blue). A cell in each field is outlined (orange) and a low GFP-FCHO1 expressing cell (arrow) and adjacent untransfected FCHO1/2-depleted cells (asterisks) are indicated. Scale bar (for E – L ): 10 μm. ( I – L ) Medial plane optical section of GFP-FCHO1 (1–889) transfected HeLa clone #64/1.E cells following a 10-min pulse of transferrin. The perimeter of a non-complemented clone #64/1.E cell is indicated (orange). DOI: http://dx.doi.org/10.7554/eLife.04137.011

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Incubation, Staining, Transfection, Labeling, Immunolabeling, Expressing

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A – H′ ) Representative single confocal optical sections of HeLa clone #64/1.E cells transiently transfected with GFP ( A and A′ ), GFP-FCHO1 (1–889) ( B and B′ ), GFP-FCHO1 EFC domain (1–275) ( C and C′ ), GFP-FCHO1 μHD (609–889) ( D and D′ ), GFP-FCHO1 (1–609) ( E and E′ ), GFP-FCHO1 (1–416) ( F and F′ ) GFP-FCHO1 (265–609) ( G and G′ ) or GFP-FCHO1 (1–889; Δ316–467) ( H and H′ ). Fixed cells were stained for AP-2 using the anti-AP-2 α-subunit mAb AP.6. Merged channel images ( A – H ) and the corresponding AP-2 α subunit channel alone ( A′ – H′ ) are shown. Transgene-dependent refashioning of the irregular 1.E cell clathrin-coated structures to a more uniformly dispersed pattern is indicated (arrows). Scale bar for all panels: 10 μm. ( I ) Cartoon diagram of the overall domain organization of FCHO1 with the relative locations of the various N-terminally-tagged truncation and deletion constructs tested shown schematically. The minimal region necessary to correct the surface clathrin morphology is boxed. ( J – J′ ) Selected confocal section of HeLa clone #64/1.E cells transfected with GFP-Sgip1 (1–514) analyzed as in ( A – H′ ). DOI: http://dx.doi.org/10.7554/eLife.04137.013

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Transfection, Staining, Construct

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A – D ). MCF-7 cells transiently transfected with either fill-length GFP-tagged FCHO1 (residues 1–889) ( A ), GFP-FCHO2 (residues 1–810) ( B ), GFP-FCHO1 EFC + linker (residues 1–609) ( C ) or GFP-FCHO1 EFC domain (residues 1–275) ( D ) were fixed and stained with an antibody (mAb AP-6) against the α subunit of the AP-2 adaptor complex (red). Forced expression of FCHO1 or FCHO2 remodels the endogenous clathrin-coated structures into more uniform arrays, as visualized by AP-2 ( A , B and C ). Although full-length GFP-FCHO1 or FCHO2 (green) concentrate at AP-2 positive surface spots, GFP-FCHO1 EFC + linker fully corrects AP-2 patterning while being diffusely associated with the plasma membrane in a featureless manner ( C ) similar to the EFC domain alone ( D ). Enlarged views of the boxed regions are shown on the lower right. Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.04137.015

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Transfection, Staining, Expressing

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A ) Muniscin–AP-2 interactions. T-Coffee generated multiple sequence alignment of the phylogenetically conserved linker region within muniscin members. Amino acid regions of Fcho1 from selected species: Homo sapiens ( Hs ; NP_001154829), Rattus norvegicus ( Rn ; XP_006252925), Python bivittatus ( Pb ; XP_007436694), Fcho2: Hs (NP_620137), Danio rerio ( Dr ; NP_001018617), Fcho2-like (Fcho2l): Aplysia californica ( Ac ; XP_005111676), the single FCHO member in Drosophila melanogaster ( Dm; NP_001097723), and Daphnia pulex ( Dp ; EFX87825), as well as Sgip1: Mus musculus ( Mm ; NP_659155) and Dr (XP_005165952) are shown with appropriate residues numbers indicated. Identical residues are highlighted in magenta, highly similar residues in pale pink and conservatively substituted amino acids in yellow. The location of mass-spectrometry authenticated phosphosites are shown (red font). ( B ) Samples of 100 μg of GST, GST-FCHO1 (316–467) or GST-ARH (180–308) immobilized on glutathione-Sepharose were incubated with rat brain cytosol, washed and samples of each supernatant (S) and pellet (P) fraction separated by SDS-PAGE. Replicate gels were either stained with Coomassie blue (left) or immunoblotted with the designated antibodies (right). The positions of the molecular mass standards (in kDa) are indicated on the left. ( C ) Schematic illustration of the overall chain and domain composition of the AP-2 adaptor complex. ( D ) Samples of 100 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–444) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays with the purified AP-2 heterameric core complex as in ( B ). The identity of the large subunit trunk polypeptides (α and β2 subunits) and the myc-tagged μ2 subunit is confirmed on immunoblots with mAb clone 8, mAb 100/1 and mAb clone 31, respectively. ( E ) Aliquots of 100 μg of GST or either 25 μg or 100 μg of GST-EPS15 (595–896) or GST-EPS15 (595–896/Δ617–636) immobilized on glutathione-Sepharose were incubated with soluble lysate from K562 cells. After washing, portions of the supernatant and pellet fractions were analyzed as in ( B ). FCHO1 antibody 1 is a mAb while antibody 2 is an affinity-purified antibody that also recognizes FCHO2 weakly. Non-specific cross-reactive bands are indicated with asterisks; reactivity of the GST-EPS15 fusion proteins with the anti-FCHO1 and anti-FCHO2 antibody preparations is also indicated with asterisks. Notice the decreased FCHO1 and FCHO2 binding upon deletion of the minimal μHD sequence within the EPS15 C-terminal region that correlates with reduced clathrin association. DOI: http://dx.doi.org/10.7554/eLife.04137.016

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Generated, Sequencing, Mass Spectrometry, Incubation, SDS Page, Staining, Purification, Western Blot, Affinity Purification, Binding Assay

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A ) Samples of ∼200 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–740) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays as in with rat brain cytosol as in . HC; heavy chain. The anti-Necap 1 antibody has extensive non-specific reactivity with the GST-fusion proteins (yellow asterisks) but, compared with the GST control, there is little loss of the Necap 1 from the supernatants after incubation with GST-muniscin linker fusions, as is seen for AP-2 subunits. Thus Necap 1 interacts only weakly with the FCHO1 linker segment. ( B ) Sample of ∼200 μg of GST, GST-FCHO1 (316–467) or GST-EPS15 (595–740) immobilized on glutathione-Sepharose were used in pull-down assays with rat brain cytosol alone or supplemented with 5 μM or 25 μM purified AP-2 α appendage. The marked recovery of the α appendage together with the GST-EPS15 fusion is not paralleled in the GST-FCHO1 linker pellets, and competition with soluble intact AP-2 is not evident. The reduced apparent abundance of the μ2 subunit in the pellet fractions from the GST-FCHO1 linker pull-down assays is due to comigration of the μ2 subunit with the GST fusion protein. ( C ) Samples of 250 μg of GST or 50 μg or 250 μg of GST-Necap 1 PHear domain (residues 1–133) immobilized on glutathione-Sepharose were incubated with K562 cell Triton X-100 lysate. After washing, aliquots of the supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE and either stained with Coomassie blue (top) or transferred to nitrocellulose in replicate. Blots were probed with affinity-purified antibodies directed against FCHO1 or FCHO2 or with a mAb (C-8) the α subunit of AP-2. FCHO1, FCHO2 and AP-2 all show dose-dependent interactions with the Necap 1 PHear domain ( , , ), although FCHO1 clearly displays the highest apparent affinity. In general, FCHO2 shows a weaker capacity to correct the clathrin distribution phenotype in HeLa clone #64/1.E cells, which is correlated with poorer binding to AP-2 and Necap 1. DOI: http://dx.doi.org/10.7554/eLife.04137.017

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Incubation, Purification, SDS Page, Staining, Affinity Purification, Binding Assay

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A – A″ ) Selected medial ( A and A′ ) or basal ( A″ ) optical sections of deconvolved confocal z -stacks collected from HeLa SS6 cells transiently transfected with Tac-FCHO1 linker (residues 265–609). Fixed cells were stained with anti-AP-2 α subunit mAb AP.6 (green) and affinity purified anti-EPS15 antibodies (red) and mounted in Hoechst 33342 to label DNA (blue) prior to imaging. Aberrant accumulation of AP-2 and EPS15 adjacent to the nucleus (arrows) in Tac-transfected cells (asterisks) correlates with smaller clathrin-coated puncta in the basal optical section of the same cell population ( A″ ). Scale bar (for A – C″ ): 10 μm. ( B – C″ ) Chosen middle ( B , B′ , C and C′ ) or maximal projection ( B″ and C″ ) optical sections of HeLa SS6 cells transfected with Tac-FCHO1 μHD (residues 609–889) or Tac-FCHO1 linker + μHD (residues 265–889) and prepared analogously to ( A–A″ ). The Tac-fused μHD produces dramatic relocalization of EPS15 ( B ), diminishing this protein in surface AP-2-positive puncta ( B ″) yet AP-2 does not relocate similarly. With ectopic expression of the FCHO linker + μHD, prominent accumulation of irregular intracellular AP-2 and EPS15 in a juxtanuclear locations again correlates with diminished surface clathrin spots, which are again relatively deficient in EPS15 because of the massive deposition of this endocytic pioneer component upon intracellular membranes. ( D – D″ ) Single ventral optical section of HeLa SS6 cells transfected with Tac-FCHO1 linker + μHD (residues 265–889) and stained with anti-Tac mAb (7 G7B6; green) and affinity-purified anti-EPS15 antibodies (red). Relocalization of EPS15 occurs only in Tac overexpressing cells (arrows), and the dose-dependent massing of EPS15 in the perinuclear region limits the amount of EPS15 present in surface clathrin-coated structures. The limiting membrane of the Tac-expressing cells is delineated (orange). Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.04137.019

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Transfection, Staining, Affinity Purification, Imaging, Expressing

Journal: eLife

Article Title: A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

doi: 10.7554/eLife.04137

Figure Lengend Snippet: ( A – F ) HeLa SS6 cells cotransfected with a mixture of either Tac-FCHO1 linker (residues 265–609) ( A – C ) or Tac-FCHO1 linker + μHD (residues 265–889) ( D – F ) and GFP-PLCδ1 PH domain encoding plasmids were fixed and stained with anti-AP-2 α subunit mAb AP.6 (red) and affinity purified anti-EPS15 antibodies (blue). The GFP fluorescence in the medial region of the Tac-linker expressing cells ( C ) represents the soluble pool of this fluorescent lipid probe adjacent to the nucleus. Note also the decrease in size and more regular arrangement of the AP-2- and EPS15-positive clathrin coated surface structures in the Tac-linker ( A ) and Tac-linker + μHD ( D ) expressing cells, identified by mislocalized deposition in a juxtanuclear position (arrows). Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.04137.020

Article Snippet: The mAbs directed against the AP-2 α subunit clone 8/Adaptin α (1:1000; 610502) and μ2 subunit clone 31/AP50 (1:500; 611350) were from BD Transduction Laboratories (San Jose, CA).

Techniques: Staining, Affinity Purification, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors *

doi: 10.1074/jbc.M113.535211

Figure Lengend Snippet: MHCI endocytosis depends on clathrin and AP-2. A, chemical structures of Pitstop-2, Pitstop 2–100, Pitnot-2, and Pitnot-2-100. B, IC50 values of Pitstop-2- or Pitstop-2-100-mediated interference with clathrin TD complex formation determined by ELISA. C, internalization of transferrin-Alexa 488 into HeLa cells is inhibited by Pitstop-2 or Pitstop-2-100 (30 μm). The amount of transferrin-Alexa 488 internalized into DMSO-treated cells was set to 100%. D, MHCI internalization into HeLa cells is inhibited by Pitstop-2 or the related compound Pitstop-2-100 (30 μm) but not by two different non-clathrin-binding control compounds. Scale bar, 10 μm. E, representative images of MHCI internalization into CHC- or AP-2(μ)-depleted HeLa cells. Cells were treated with either DMSO or Pitstop-2. Scale bar, 10 μm. F, quantification of data shown in D (n = 3 independent experiments). The amount of MHCI internalized into DMSO-treated cells was set to 100%. G, quantification of MHCI internalization into clathrin (CHC k.d.)- or AP-2(μ)-depleted (μ2 k.d.) cells treated with DMSO or Pitstop-2 as shown in E. The amount of MHCI internalized into DMSO-treated control cells was set to 100% (n = 3 independent experiments; *, p < 0.05; **, p < 0.005; ***, p < 0.0005). H, representative images of MHCI surface levels of CHC- or AP-2(μ)-depleted HeLa cells. Cells were treated with either DMSO or Pitstop-2. Scale bar, 10 μm. I, quantification of clathrin (CHC) or AP-2(α) levels in cells treated with siRNA against AP-2(μ) or CHC or mock transfected (n = 3 independent experiments).

Article Snippet: For Western blot experiments, antibodies were used against clathrin heavy chain (TD1, homebrew), AP-1 (γ1-adaptin, BD Transduction, 610386), AP-2 (α-adaptin AC1M11, Dianova, MA3-061), AP-3 (μ3-adaptin p47, BD Transduction, 610901), EGF receptor (D38B1, Cell Signaling, 4267S), and β-actin (ac-15, Sigma, A-5441).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors *

doi: 10.1074/jbc.M113.535211

Figure Lengend Snippet: Pitstop-2-mediated inhibition of CCP dynamics has no major effects on general plasma membrane mobility. A, time lapse series of cells stably expressing AP-2(σ2)-eGFP were collected after clathrin HC depletion or mock treatment using live cell TIRF microscopy. Cells were imaged for 2 min at 37 °C in presence of 30 μm Pitstop-2 or DMSO (0.1%). AP-2(σ2)-eGFP distribution is unaffected by clathrin HC knockdown (CHC k.d.) or application of Pitstop-2, respectively. Kymographs depict stalled AP-2(σ2)-eGFP dynamics in absence of clathrin HC or after Pitstop-2-mediated inhibition of clathrin TD. Scale bar, 10 μm. B, FRAP analysis of HeLa expressing GPI-eGFP and either left untreated or treated with 0.1% DMSO or with 30 μm of the indicated compounds (Pitstop-2, Pitstop-2-100, Pitnot-2, or Pitnot-2-100; see Fig. 1A). 60 s after bleaching, Pitstop-2-treated cells show similar recovery to DMSO-treated controls. Scale bar, 10 μm. C, quantitative analysis of fluorescence recovery as shown in B. Graphs represent the relative fluorescence recovery normalized to the fluorescent intensity before bleaching (n = 3 independent experiments; *, p < 0.05).

Article Snippet: For Western blot experiments, antibodies were used against clathrin heavy chain (TD1, homebrew), AP-1 (γ1-adaptin, BD Transduction, 610386), AP-2 (α-adaptin AC1M11, Dianova, MA3-061), AP-3 (μ3-adaptin p47, BD Transduction, 610901), EGF receptor (D38B1, Cell Signaling, 4267S), and β-actin (ac-15, Sigma, A-5441).

Techniques: Inhibition, Stable Transfection, Expressing, Microscopy, Fluorescence

Journal: Cell Death & Disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: A RNA-Sequencing of different melanoma cell lines (SbCl2, WM3211, WM1158, WM793, WM9) depicting a heterogenic expression pattern of the 5 AP2-isoforms. RNA-Seq read counts were normalized to library size. B Immunohistochemical stainings of primary melanoma tumor (PT) or metastasis (Met). AP2α and AP2γ stainings are published by the human protein atlas (AP2α: https://www.proteinatlas.org/ENSG00000137203-TFAP2A/pathology/melanoma AP2γ: https://www.proteinatlas.org/ENSG00000087510-TFAP2C/pathology/melanoma ; accessed on 08/09/2023, 11:51 am). AP2ε was stained with a specific anti-AP2ε-antiserum, established in our group . a, b Image sections depicting high nuclear staining of AP2α in primary tumor and metastasis. c, d Image sections showing weak nuclear staining of AP2γ in primary tumor and low staining in metastasis. e, f Image sections depicting low nuclear staining in primary tumor and only low staining in approximately 5% of the cells in the metastasis for AP2ε. (Scale bar: 200 μm). C Kaplan-Meier survival curve analysis for AP2α, AP2γ, AP2ε in malignant melanoma was performed using the TCGA-derived datasets deposited on the ProteinAtlas database. Survival analysis was performed computationally applying log-rank testing. D Expression analysis for AP2α, AP2γ, and AP2ε published on OncoDB ( https://oncodb.org/index.html ) comparing normal skin tissue (non-tumor) and malignant melanoma (MM) tissue. Data information: Data are represented as mean ± SEM; * p < 0.05 (Two-tailed Student’s t-test).

Article Snippet: AP2α and AP2γ stainings are published by the human protein atlas (AP2α: https://www.proteinatlas.org/ENSG00000137203-TFAP2A/pathology/melanoma AP2γ: https://www.proteinatlas.org/ENSG00000087510-TFAP2C/pathology/melanoma ; accessed on 08/09/2023, 11:51 am).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Staining, Derivative Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: A Mel Im cell growth over 7 days (Scale bar = 200 µm). B Cell cycle analysis of Mel Im FUCCI in CIB and MG, respectively. C Mel Im migration in CIB during cultivation (Scale bar: 200 µm). D mRNA-expression analysis for AP2α, AP2γ, and AP2ε in MG and CIB ( n = 3 ). E Immunohistochemical staining for AP2ε in Mel Im cells bioprinted in CIB and MG. F Relative luciferase activity in Mel Im cells transfected with an AP2-Luciferase construct and afterwards cultivated in MG or CIB ( n = 3 ). G Relative luciferase activity in Mel Im cells transfected with an AP2-Luciferase construct after transfection of siRNA against AP2ε and control-siRNA, respectively. After transfection cells were cultivated in MG ( n = 3 ) or CIB ( n = 4 ). H mRNA-expression analysis for AP2ε after cultivating the melanoma cell lines Mel Im and 501 Mel under hypoxic conditions ( n = 3 ). I Immunofluorescence images of AP2ε and HIF1-α or Ki-67 in the human melanoma tissue samples (Magnification: 40x). Data information: All data from at least three independent experiments are represented as mean ± SEM; * p < 0.05; ns : not significant; (Two-tailed Student’s t-test) ( D , F , G ); (Scale bars ( A , C , E , I ): 10 µm).

Article Snippet: AP2α and AP2γ stainings are published by the human protein atlas (AP2α: https://www.proteinatlas.org/ENSG00000137203-TFAP2A/pathology/melanoma AP2γ: https://www.proteinatlas.org/ENSG00000087510-TFAP2C/pathology/melanoma ; accessed on 08/09/2023, 11:51 am).

Techniques: Cell Cycle Assay, Migration, Expressing, Immunohistochemical staining, Staining, Luciferase, Activity Assay, Transfection, Construct, Control, Immunofluorescence, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Transcription factor activating enhancer-binding protein 2ε (AP2ε) modulates phenotypic plasticity and progression of malignant melanoma

doi: 10.1038/s41419-024-06733-3

Figure Lengend Snippet: A Representative AP2ε immunohistochemical analysis of lung metastasis from Tg(GRM1) and AP2ε -/- /Tg(GRM1) mice. a, b, c, d Red arrows depicting AP2ε positive nuclear stainings. Number of disseminated cells per field of view of 3 mice per genotype in 10 sections per mice have been counted ( n = 3). (Scale bar: 200 µm). B Migratory behavior of AP2ε -/- /Tg(GRM1) cells compared to Tg(GRM 1 ) using the Boyden chamber model. ( n = 3). C Attachment analyses by using the xCELLigence system of Tg(GRM1) and AP2ε -/- /Tg(GRM1) for cells from metastatic lung tissue (Delta Cell index = relative change in measured impedance to represent cell status) ( n = 3). D Migratory behavior of Mel Im cells transfected with the AP2ε-overexpression plasmid compared to control vector pCMX using the Boyden chamber model. ( n = 3). E Relative luciferase activity in primary AP2ε -/- /Tg(GRM1) and Tg(GRM1) cells, respectively, transfected with an AP2-Luciferase construct and afterwards cultivated in CIB ( n = 3 ). F Spheroid outgrowth assay depicting reduced migratory activity in AP2ε -/- /Tg(GRM1) spheroids. Quantification of the distance of outgrowth in µm. G Embedded AP2ε -/- /Tg(GRM1) and Tg(GRM1) spheroids after 14 days in MG. White arrows depicting outgrown cells from the Tg(GRM1) spheroid. Protrusion lengths of 9 spheroids per condition have been counted after 10 days in MG ( n = 3). Data information: All data from at least three independent experiments are represented as mean ± SEM; * p < 0.05 (two-tailed Student’s t-test) ( B , C , D , E ); For A and G an unpaired t-test with Welch’s correction was applied; number of experimental animals ( A ) n = 3.

Article Snippet: AP2α and AP2γ stainings are published by the human protein atlas (AP2α: https://www.proteinatlas.org/ENSG00000137203-TFAP2A/pathology/melanoma AP2γ: https://www.proteinatlas.org/ENSG00000087510-TFAP2C/pathology/melanoma ; accessed on 08/09/2023, 11:51 am).

Techniques: Immunohistochemical staining, Transfection, Over Expression, Plasmid Preparation, Control, Luciferase, Activity Assay, Construct, Two Tailed Test