Journal: Nature Communications

Article Title: Structural heterogeneity of the ion and lipid channel TMEM16F

doi: 10.1038/s41467-023-44377-7

Figure Lengend Snippet: a Membrane topology of a TMEM16F subunit. The transmembrane helices (numbered from 1 to 10) important for ion/lipid translocation and subunits dimerization are highlighted in green (pore domain) and sky-blue (dimerization domain), respectively. NCD, N-terminal cytosolic domain. b Cartoon showing the pore domain architecture of TMEM16F. The permeation pathway is gated by Ca 2+ ions (red-filled circles) and can function as an ion channel (middle panel), and/or lipid scramblase (right panel). c Membrane patches isolated from NG108-15 cells (dotted outline) were identified by AFM imaging ( n = 47 independent topographic images) before unfolding experiments commenced. Cross-sectional analysis along the white dotted line is shown in i . d Schematics illustrating the unfolding process of a TMEM16F protomer. The AFM probe is first brought into contact with a TMEM16F to aid physisorption of the NCD to the AFM tip. Afterwards, the probe is retracted, thereby applying a mechanical force unfolding the protein, and an F-D curve is recorded. e Recombinant TMEM16F was engineered to bear the N2B fingerprint and the GFP polypeptide at the N- and C-terminus, respectively (left panel). A representative unfolding of the N-N2B-16F construct from the N-terminus is shown at the right. The double-headed arrow designates the unfolding of the N2B segment. The C-terminal GFP allows individuation of successfully transfected cells and location of TMEM16F on the cell membrane. f Representative normalized currents recorded from inside-out membrane patches from HEK293 cells expressing WT-16F. The upper panel shows currents recorded at the holding potential of +60 mV upon a Ca 2+ concentration jump from nominally 0 to 1 mM. The lower panel shows currents activated by voltage ramps from −40 mV to +40 mV in the presence of 1 mM CaCl 2 in symmetrical 140 mM NaCl (Sym NaCl) or 14 mM intracellular NaCl solutions (Low NaCl). g As in f but for the N-N2B-16F fusion construct. h Examples of F-D spectra corresponding to the mechanical unfolding of WT-16F from the N-terminus in the absence of Ca 2+ . Arrowheads designate commonly observed unfolding intermediates. i Superposition of representative WT-16F (black), N-N2B-16F (ochre), and purified TMEM16F (blue lines) reconstituted in proteoliposomes.

Article Snippet: The cells were incubated with specific primary antibodies at a ratio of 1: 400 (TMEM16B Cat# 20647-1-AP, Proteintech Euro; TMEM16F Cat# ACL-016, Alomone Labs) or 1x PBS (Control) at 4 °C overnight, followed by rinsing with pre-cold 1xPBS 3 times.

Techniques: Membrane, Translocation Assay, Isolation, Imaging, Recombinant, Construct, Transfection, Expressing, Concentration Assay, Purification

Journal: Nature Communications

Article Title: Structural heterogeneity of the ion and lipid channel TMEM16F

doi: 10.1038/s41467-023-44377-7

Figure Lengend Snippet: a Superposition of 78 F-D curves from recombinant N-N2B-16F pulled in the absence of Ca 2+ . Only the unfolding of TMEM16F monomers is shown. b Violin plots of the calculated unfolding work for the N-N2B-16F ( n = 78; ochre hue), WT-16F ( n = 101; gray hue) and purified murine TMEM16F reconstituted into liposomes (PR-16F, n = 68; blue hue). Average work and standard deviations correspond to 16.4 ± 7.5, 18.4 ± 8.4, and 20.4 ± 8.7 aJ for N-N2B-16F, WT-16F, and PR-16F constructs, respectively. A multimodal distribution with two prominent populations referred to as low (LW) and high unfolding work (HW) was observed in all constructs (arrowheads). c Medium-resolution AFM image of reconstituted TMEM16F protein ( n = 4 independent AFM experiments). Individual TMEM16F exposing either extracellular or intracellular side are clearly discernible (white and red arrowheads, respectively). d High-resolution images of two representative TMEM16F dimers exposing the intracellular face. Compact (PR-16F(cd), left; n = 5 independent molecules/experiments) and loose (PR16F(ld), right; n = 5 independent molecules/experiments) configurations with the two protomers further apart were observed. Both molecules were imaged within 24 h with the same HS-AFM scanner, and image area dimensions. e Height profile analysis along the white (dp) and blue (cd and ld) dashed lines (see insets). In the loose configuration, a ~ 1 nm membrane depression is often observed in-between the TMEM16F protomers.

Article Snippet: The cells were incubated with specific primary antibodies at a ratio of 1: 400 (TMEM16B Cat# 20647-1-AP, Proteintech Euro; TMEM16F Cat# ACL-016, Alomone Labs) or 1x PBS (Control) at 4 °C overnight, followed by rinsing with pre-cold 1xPBS 3 times.

Techniques: Recombinant, Purification, Liposomes, Construct, Membrane

Journal: Nature Communications

Article Title: Structural heterogeneity of the ion and lipid channel TMEM16F

doi: 10.1038/s41467-023-44377-7

Figure Lengend Snippet: a Endogenous TMEM16F channel occasionally unfolds in tandem. Three main unfolding patterns (labeled 1–3) were observed, revealing differences in inter-subunits mechanical interactions. b , c The hypothesized dimerization models (C-C, C-N, and N-N) were identified by concatenating to WT-16F unfolded dimers (black traces) representative spectra from TMEM16F monomers wherein the N2B tag was conjugated either to the N- (N-N2B-16F in red colors) or C-terminal end (C-N2B-16F in cyan colors). Sketches of the engineered constructs and their putative unfolding polarity are shown at the bottom of the unfolded spectra. (*) denotes the inferred dimerization interface. Gray double-headed arrow designates the unfolding of the N2B segment. d Schematics depicting the three proposed dimerization models. In C-C model protomers interaction is mediated by C-terminal domains (1); in C-N model by C- and N-terminal domains (2); and in N-N model by N-terminal domains (3). Red and blue colors denote the two TMEM16F subunits. e HS-AFM images (from Supplementary Movie 1) of a TMEM16F dimer from the intracellular side. Monomers display significant relative motion, swinging away (red arrows) and back (green arrows) amid compact-symmetric dimers and loose-asymmetric arrangements. Similar subunit motions (sliding and/or rotation, see main text) were observed in three independent experiments. f The observed dimer configurations can be qualitatively sorted into three structural classes referred to as cryo-EM like (em), slid (si), and open/rotated (o/r). Three examples for each class (i–iii) are reported. g Simulated AFM and automatized fitting procedures were used to reconstruct the TMEM16F quaternary structure from the deposited TMEM16F cryo-EM file (pdb 6P46, see Methods). Simulated AFM images (left) and corresponding molecular structures (right) obtained after fitting are shown for the three main structural classes (em, si, o/r). Similarity scores (r) are reported. S 1 (red) and S 2 (blue) designate the two protomers. h Cryo-EM-like (em) and open/rotated (o/r i–iii) configurations were superimposed to highlight rotational motions of subunits. Subunit S 2 (in blue) was used as a reference for structures registration. A clam-shell mechanism describes sufficiently well the observed conformational dynamics. Em and o/r structural classes are filled in white and different red shades colors, respectively. The o/r class shows some structural fluctuations (compare i–iii).

Article Snippet: The cells were incubated with specific primary antibodies at a ratio of 1: 400 (TMEM16B Cat# 20647-1-AP, Proteintech Euro; TMEM16F Cat# ACL-016, Alomone Labs) or 1x PBS (Control) at 4 °C overnight, followed by rinsing with pre-cold 1xPBS 3 times.

Techniques: Labeling, Construct, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural heterogeneity of the ion and lipid channel TMEM16F

doi: 10.1038/s41467-023-44377-7

Figure Lengend Snippet: a TMEM16F unfolding in the absence of Ca 2+ . Left panel: Superposition of 78 F-D curves from unfolding recombinant N-N2B-16F obtained in the presence of 1 mM EGTA. Worm-like chain (WLC) curves corresponding to average contour lengths (Lcs) of each force peak are overlayed (gray curves). Right panel: Contour length (Lc) histogram of all force peaks detected in the F-D curves shown in the density plot at the left. Histogram was fitted with multiple Gaussian providing mean Lcs (indicated at the top of each Gaussian distribution and WLC curve) and force peak probabilities (see text and Supplementary Table ). The six major peak classes were occasionally preceded by less defined unfolding events (arrowhead). dtc., detachment peak. b Representative F-D spectra obtained in the absence of Ca 2+ . Schematic representations of hypothesized interactions between the transmembrane helices are shown below. Dots indicate the approximate location of the force peaks that do or do not undergo major changes upon Ca 2+ binding (dashed and solid symbols, respectively). c , d As in a but in the presence of 2 mM Ca 2+ ( n = 70). e Time-lapse analysis of TMEM16F subunits height (S and S 2 in green and gray colors, respectively) and changes in lipid volume (∆VL, black color). Upon Ca 2+ injection, subunit S 2 moves away from the membrane plane by ~1 nm and concurrently remodeling of lipid bilayer (LR) in and around the TMEM16F dimer is observed (black symbols in plot e and dotted white outline in insets ii-iv). These morphological changes in the membrane around TMEM16F dimers are possibly related to TMEM16F lipid scrambling and were observed in two independent experiments. In agreement with the large variability in functional measurements reported in Fig. such effect was not observed in all molecules/experiments. f Height distributions for S (left panel) and S 2 (right panel) suggesting independent subunits motion and two main states at around ~2.8 and ~3.7 nm height. Sky blue and salmon colors refer to distributions observed in the presence of EGTA and Ca 2+ , respectively.

Article Snippet: The cells were incubated with specific primary antibodies at a ratio of 1: 400 (TMEM16B Cat# 20647-1-AP, Proteintech Euro; TMEM16F Cat# ACL-016, Alomone Labs) or 1x PBS (Control) at 4 °C overnight, followed by rinsing with pre-cold 1xPBS 3 times.

Techniques: Recombinant, Binding Assay, Injection, Membrane, Functional Assay

Journal: Nature Communications

Article Title: Structural heterogeneity of the ion and lipid channel TMEM16F

doi: 10.1038/s41467-023-44377-7

Figure Lengend Snippet: a , b Inside-out excised membrane patches from transiently transfected HEK293 cells expressing TMEM16F were recorded in symmetrical NaCl (140 mM, a ) and lower intracellular NaCl (14 mM, b ) solutions. Their IV relations were determined via voltage ramps from −80 mV to +100 mV. Currents were activated by 1 mM CaCl 2 and normalized to the current at +100 mV. c , d The same as a , b but for TMEM16B. e Comparison of the changes of rectification for TMEM16F (black) and TMEM16B (gray) currents. Rectification was calculated as the ratio between currents measured at +60 and −60 mV (|I +60 /I −60 |) and normalized to the average rectification value µ (the average current at –60 mV is –266 ± 199 pA for TMEM16F, −430 ± 286 pA for TMEM16B and –0.2 ± 2 pA for not transfected cells (nt); at +60 mV is 673 ± 455 pA for TMEM16F, 293 ± 221 pA for TMEM16B and –0.6 ± 3 pA for nt cells, n = 18 for 16F, n = 7 for 16B an n = 6 for nt). f Comparison of the shift of reversal potentials after the replacement of 140 mM NaCl with 14 mM NaCl for TMEM16F and TMEM16B ( n = 19 for 16F and n = 7 for 16B). In panels 5e,f mean values +/- standard deviation are overlaid to individual data points. g , h Violin plots of unfolding work of Ca 2+ -bound N-N2B-16F ( n = 70, average W 12.0 aJ, g) and N-N2B-16B ( n = 38, average W 8.8 aJ, h).

Article Snippet: The cells were incubated with specific primary antibodies at a ratio of 1: 400 (TMEM16B Cat# 20647-1-AP, Proteintech Euro; TMEM16F Cat# ACL-016, Alomone Labs) or 1x PBS (Control) at 4 °C overnight, followed by rinsing with pre-cold 1xPBS 3 times.

Techniques: Membrane, Transfection, Expressing, Comparison, Standard Deviation

Journal: BMC Complementary Medicine and Therapies

Article Title: Integrated proteomics and metabolomics analysis of lumbar in a rat model of osteoporosis treated with Gushukang capsules

doi: 10.1186/s12906-022-03807-7

Figure Lengend Snippet: Differential proteins and metabolites enriched by the common metabolic pathways in GSK/OVX and OVX/SHAM. * represents a protein or metabolite with differential expression trends between GSK/OP and OP/SHAM

Article Snippet: Anti-Slc1a3 rabbit polyclonal antibody [EPR12686] was from Abcam Plc ((Cambridge, USA), anti-Aldh3b1 rabbit polyclonal antibody [TA323583S] was from OriGene Technologies, Inc. (Rockwell, USA), anti-Gstz1 rabbit polyclonal antibody [No.Ag6676], anti-Cant1 rabbit polyclonal antibody [No.12164–1-AP], anti-Bid rabbit polyclonal antibody [No.10988–1-AP] were from Proteintech Group, Inc. (Rosement, USA).

Techniques: Quantitative Proteomics, Activation Assay