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

Abstract

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.

Fig. 1

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

Fig. 2

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, Ca²⁺ 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)

Fig. 3

The myosin VI-binding region of CLCa forms an α-helix that interlocks with the helices of myosin VI. a 2D ¹H, ¹⁵N-HSQC spectrum of 0.78 mM ¹⁵N-labeled myosin VI^1050–1131 alone (blue) and in the presence of equimolar unlabeled CLCa^46–61 (orange). Notable changes in NMR signals following addition of CLCa are indicated with arrows and labeled with the assigned amino acid residue from myosin VI. b On the previously solved structure of myosin VI^1050–1131 (PDB 2N12), amino acids with CSPs greater than the mean or 1 SD above the mean are indicated in orange or red, respectively. CSPs were calculated according to the definition CSP = [(0.2ΔδN)² + (ΔδH)²]^1/2 where ΔδN and ΔδH represent chemical shift differences for the amide nitrogen and proton of each residue, respectively. Residues without amide protons (P1051, P1055, and P1070) are indicated in gray, and those at the beginning and end of each helix are labeled. c Superimposition of the 20 lowest energy structures calculated for myosin VI^1050–1131 in complex with CLCa^46–61. CLCa is shown in yellow; myosin VI is colored cyan except for the isoform-specific α2 helix, which is highlighted in orange. The orientation of the structures is identical to b. d Ribbon representation of the structure of myosin VI bound to CLCa^46–61 using the same color scheme and orientation as in c. e Ribbon representation as in d, expanded and rotated about the CLCa helix to highlight the α-helical structure of CLCa^46–54 and interactions involving the C-terminal amino acids of the CLCa peptide. Sidechain heavy atoms of the highlighted residues are included. Note that the C-terminal portion of the CLCa fragment remains in close contact with myosin VI and the CLCa helix. f Selected regions from a 3D ¹³C NOESY experiment acquired on 0.4 mM ¹³C, ¹⁵N-labeled CLCa^46–61 and equimolar unlabeled myosin VI^1050–1131. NOE interactions involving the C-terminal portion of CLCa (A59, P60, and G61) are shown. Intramolecular and intermolecular interactions are labeled in red and black, respectively

Fig. 4

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. 3e 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

Fig. 5

CLCa I54D is a selective myosin VI-impaired mutant. a Co-immunoprecipitation experiment using lysates from Caco-2 reconstituted cell lines. Cells depleted of endogenous CLCs or mock treated are kept in confluency for 7 days, lysed, and RFP-CLCa wild-type and I54D were immunoprecipitated from 1 mg of lysates. IP and IB as indicated. b Representative confocal micrographs of CLCa wild-type and I54D localization in Caco-2 cysts. Staining with the indicated apical–basal polarity markers are shown. Scale bar, 100 µm. c Upper panel, representative brightfield pictures of Caco-2 cysts generated using the indicated isogenic cell lines. Scale bar, 1 mm. Clathrin-coated structures present at the apical surface of the Caco-2 cysts were counted and classified as shallow, omega/constricted, or elongated according to their morphology. Left panel, representative EM images of the different morphology of CCPs. Scale bar, 100 nm. Right panel, distribution of clathrin-coated structures in the different isogenic cell lines expressed as percentage among the different classes and calculated on three independent experiments. Between 94 and 125 cellular profiles of well-polarized cells for each line were imaged. Number of clathrin-coated structures counted in total are reported as n = x. See also Supplementary Fig. 11d

Fig. 6

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 2. 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