gst myosin vi  (Addgene inc)

Journal: Nature Communications

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

doi: 10.1038/s41467-019-12855-6

Figure Lengend Snippet: CLCa is a direct and specific interactor of myosin VI long in triskelia and clathrin cages. a Scheme of the myosin VI highlighting the region involved in clathrin binding (amino acids 998–1131 of the long isoform). Long and short isoforms are reported together with the domains and motifs previously identified, including IQ motif, 3HB (three-helix bundle), SAH (single α-helix), MIU (motif interacting with ubiquitin), AS (alternative splicing region), and MyUb (myosin VI ubiquitin-binding domain). In orange is represented the alternatively spliced region codifying for the α2-linker . b Pull-down assay with GST-CLCa and CLCb full-length and cleaved and purified fragments spanning amino acids 998–1131 of long and short myosin VI isoforms. Glutathione sepharose beads coupled to GST and GST-tagged proteins were incubated with myosin VI 998–1131 . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c Pull-down assay using the long and short GST-myosin VI 998–1131 constructs and brain lysates (500 μg) obtained from the indicated mouse strains. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and transferred to a nitrocellulose membrane. Immunoblot (IB) was performed with anti-clathrin heavy-chain antibody. Ponceau detect equal loading of GST proteins. d IB of the brain lysates used in ( c ), as indicated. e Co-sedimentation assay. Equimolar (1.5 μM) amount of myosin VI 998–1131 and clathrin cages were incubated at 4 °C for 45 min in the presence of detergent (0.1% Triton X-100) and then pelleted by ultracentrifugation. Precipitated proteins were dissolved in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. CLCs* indicates the various CLC proteins. Note that in the native cages CLCs (CHC-CLCab) run at different molecular weight (mw) as they are from pig brain while the human CLCs used for reconstitution are bacterially produced and cleaved from GST

Article Snippet: GST-tagged myosin VI and CLCa were expressed separately in E. coli BL21 (DE3) cells (Thermo Fischer Scientific) at 17 °C overnight upon reaching an OD 600 value of 0.5–0.6 by induction with 0.4 mM IPTG.

Techniques: Binding Assay, Pull Down Assay, Purification, Incubation, SDS Page, Staining, Construct, Western Blot, Sedimentation, Molecular Weight, Produced

Journal: Nature Communications

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

doi: 10.1038/s41467-019-12855-6

Figure Lengend Snippet: CLCa:myosin VI long interact with sub-micromolar affinity. a Domain structures of CLCa and CLCb. CON conserved Hip-binding region, Hsc70 unique region in CLCa that stimulates Hsc70 activity in vitro, Ca 2+ EF-hand domain that binds calcium, CHC binding clathrin heavy chain binding region, CBD calmodulin-binding domain. Sequence conservation between the two proteins is reported below. Each line represents one amino acid, black line indicates identity. Lower panel, scheme of the selected constructs used in ( b ) together with the sequence of the three overlapping 5-carboxyfluorescein (5-FAM)-conjugated CLCa peptides used for FP analysis in ( c ). b Pull-down assay with GST-CLCa and CLCb full length and the indicated fragments of CLCa immobilized on glutathione sepharose beads and incubated with the purified fragment spanning amino acids 998–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. c FP assay using the three peptides shown in ( a ) and the purified fragment spanning amino acids 998–1131 of myosin VI long . Dissociation constants with their respective 95% confidence interval (CI) are reported in the table at the bottom. Graph is representative of three independent experiments used to calculate K d and CI. d FP assay using peptide 46–61 of CLCa and the indicated fragments of long and short myosin VI isoforms. Graph, K d , and CI as for ( c )

Article Snippet: GST-tagged myosin VI and CLCa were expressed separately in E. coli BL21 (DE3) cells (Thermo Fischer Scientific) at 17 °C overnight upon reaching an OD 600 value of 0.5–0.6 by induction with 0.4 mM IPTG.

Techniques: Binding Assay, Activity Assay, In Vitro, Sequencing, Construct, Pull Down Assay, Incubation, Purification, SDS Page, Staining, FP Assay

Journal: Nature Communications

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

doi: 10.1038/s41467-019-12855-6

Figure Lengend Snippet: A hydrophobic pocket formed by myosin VI encompasses residues I54 and L55 of CLCa. a Ribbon representation of the myosin VI:CLCa binding interface in the orientation of Fig. with key sidechain heavy atoms displayed. Myosin VI α4 residues R1117, V1120, Y1121, and W1124 (blue) interact with CLCa E50, A51, I54, and L55 (yellow). CLCa L55 also interacts with myosin VI α2 residues, with M1058 and M1062 (orange) observable in this view. At the edge of the hydrophobic pocket lies a hydrogen bond between CLCa E50 and the myosin VI indole group. Sulfur and nitrogen atoms are in yellow and indigo, respectively. b As in a but rotated about the CLCa helix to highlight interactions involving the myosin VI isoform-specific α2 helix, especially P1055 and A1059. c Enlarged representation of the region containing myosin VI R1117 to highlight intramolecular and intermolecular hydrogen bonds. The guanidine group of R1117 forms hydrogen bonds to the backbone and sidechain carboxyl groups of myosin VI S1087 and E1113, respectively, and to the backbone carboxyl group of CLCa D56. This view is similar to that of a but rotated about the sidechain of R1117. In a , c , a dashed yellow line is used to indicate a hydrogen bond with oxygen and nitrogen atoms in red and indigo, respectively. d Sequence alignment of CLCa 46–61 with the corresponding region of CLCb. Asterisks indicate residues not conserved between isoforms. In yellow are amino acids putatively responsible for the selective binding. e Pull-down assay using the indicated GST-myosin VI 1050–1131 mutant constructs and lysates (1 mg) from HEK293T cells. After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and IB was performed with the anti-CLCa antibody (×16). Ponceau detects equal loading of GST proteins. Representative image of three independent experiments is shown. f GST pull-down assay using the indicated CLCa constructs and purified fragment spanning amino acids 1050–1131 of myosin VI long . After washes, bound proteins were eluted in Laemmli-buffer, resolved through SDS-PAGE, and stained with Coomassie. Bottom panel, quantitation of three independent experiments. Data are expressed as percentage of binding with respect to input and normalized for the amount of GST proteins used in each pull-down. Error bars represent s.d. *** P < 0.001 by two-tailed T test

Article Snippet: GST-tagged myosin VI and CLCa were expressed separately in E. coli BL21 (DE3) cells (Thermo Fischer Scientific) at 17 °C overnight upon reaching an OD 600 value of 0.5–0.6 by induction with 0.4 mM IPTG.

Techniques: Binding Assay, Sequencing, Pull Down Assay, Mutagenesis, Construct, SDS Page, Purification, Staining, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension

doi: 10.1038/s41467-019-12855-6

Figure Lengend Snippet: Myosin VI and Hip1R are mutually exclusive binders of CLCa. a Binding regions of myosin VI and Hip1R on CLCa. b Competitive binding assay. Bacterially purified His-Hip1R coiled-coil region spanning residues 346–655 (2.5 µM) pre-incubated with GST-CLCa full-length protein (1 µM) for 1 h at 4 °C was mixed with increasing amounts of myosin VI 1050–1131 as indicated. Bottom panel, Coomassie staining. Lower panels, 1/10 of the elutant was loaded for immunoblotting with the indicated antibodies. c As in b but using GST-CLCa I54D mutant. d ITC experiments with the indicated proteins. The integrated heat and raw plots are reported. Equilibrium dissociation constants ( K d ) obtained by the fitting are indicated below. Relevant ITC measurements are reported in Table . e A putative model of clathrin-coated pit fission in polarized tissue. Once CCP passes the endocytic checkpoint, it undergoes a maturation process mediated by PIP2 turnover and the activity of several endocytic accessory factors (e.g., Hip1R). (1) As the bud expands, myosin VI is recruited to CCPs by CLCa and disengages Hip1R, which preferentially associates with epsin at the CCP edge. Actin anchoring and polymerization is thereby restricted to the neck of the invaginating pit where Hip1R along with epsin provide a link between actin nucleation and the CCP. Alternatively, formation of a complex between epsins and Hip1R at the neck would recruit Hip1R from the coat, exposing the myosin VI-binding site on CLCa and causing recruitment of myosin VI to the CCP. (2) Myosin monomer bound to CLCa anchors the CCP to the actin meshwork engaged in retrograde flow and facilitates movement of the vesicle into the cytoplasm. After dimerization induced by Dab2 or oligomerization, myosin VI becomes a processive motor that walk toward the minus end of actin filaments providing mechanical force to oppose membrane tension in polarized tissues and promoting the final dynamin-mediated fission step (not depicted). Future experiments are needed to test this model. In the myosin VI representation, the boxing glove and white cuff indicate the motor domain and single IQ motif, respectively, while the cargo-binding tail, which includes the clathrin-binding domain, is depicted as a green sphere; the black squiggly connecting region includes the 3HB and SAH

Article Snippet: GST-tagged myosin VI and CLCa were expressed separately in E. coli BL21 (DE3) cells (Thermo Fischer Scientific) at 17 °C overnight upon reaching an OD 600 value of 0.5–0.6 by induction with 0.4 mM IPTG.

Techniques: Binding Assay, Competitive Binding Assay, Purification, Incubation, Staining, Western Blot, Mutagenesis, Activity Assay