The mechanical unfolding of proteins is a cellular mechanism for force transduction with potentially broad implications in cell fate. Despite this, the mechanism by which protein unfolding elicits differential downstream signalling pathways remains poorly understood. Here, we used protein engineering, atomic force microscopy, and biophysical tools to delineate how protein unfolding controls cell mechanics. Deleted in liver cancer 1 (DLC1) is a negative regulator of Ras homolog family member A (RhoA) and cell contractility that regulates cell behaviour when localised to focal adhesions bound to folded talin. Using a talin mutant resistant to force-induced unfolding of R8 domain, we show that talin unfolding determines DLC1 downstream signalling and, consequently, cell mechanics. We propose that this new mechanism of mechanotransduction may have implications for a wide variety of associated cellular processes.
The smAFM experiments were carried out according to protocols described previously [8]. The talin fragment polyprotein constructs, including flanking I27, were synthesised and cloned into the pFN18a vector. The polyproteins were expressed in Escherichia coli BL21-CodonPlus (DE3) -RILP competent cells, using the T7 promoter present in the plasmid. Protein expression was induced with IPTG when the culture reached an OD600 nm of 0. 6. Cells were lysed with lysozyme and sonication before protein purification with Ni-NTA beads in a batch process. The eluted proteins were analysed for purity with SDS-PAGE. Final concentration of protein used for experiments was 1–10 μg/ml. Glass coverslips were functionalised with the chloroalkane ligand to HaloTag as previously described [32]. The glass coverslips were first cleaned using Helmanex III (1% in water), acetone, and ethanol washes. The surfaces were then prepped with O2 plasma cleaning for 15 min. Surfaces were then silanised using (3-aminopropyl) trimethoxysilane diluted to 1% in ethanol. Surfaces were then washed with ethanol and then dried with N2. These amine-functionalised surfaces were then incubated with 10 mM succinimidyl-[ (Nmaleimidopropionamido) tetracosaethyleneglycol] ester (SMPEG24, Thermo) diluted in 100 mM borax buffer (pH 8. 5) for 1 h. The final step involved incubating the surfaces overnight with 10 mM HaloTag Thiol O4 ligand in the same buffer. The surfaces were quenched 50 mM 2-mercaptoethanol in water. We used a commercial AFS-1 from Luigs & Neumann, GmbH, based on a device developed at the Fernandez Lab, Columbia University [33]. The cantilevers used were gold-coated OBL-10 levers from Bruker. The spring constants varied between 4–10 pN/nm as measured by equipartition theorem with the appropriate adjustments for cantilever geometry. Around 20 μl of protein solution was incubated on functionalised coverslips for 30 min prior to the experiments to allow for HaloTag binding. The cantilever was pressed into the surface with a force of approximately 300 pN to bind the cantilever to the polyprotein. Force extension experiments were conducted at 400 nm/s retraction rate. Data analysis was carried out using Igor Pro (Wavemetrics), for which the wormlike chain model was applied. The following structures from RCSB Protein Data Bank were used as the protein models for the individual talin rod subdomains: R8 (id 5FZT residues 1451–1588), R8-DLC1 (id 5FZT talin residues 1451–1588 and DLC1 residues 467–489), and R7R8 (id 2X0C). The talin structures were modified in PyMOL to produce the desired constructs. MD and SMD simulations were performed using Gromacs ver 2016. 1 [34,35] at the Sisu supercomputer, CSC, Finland. The CHARMM27 force field [36] and explicit TIP3P water model [37] in 0. 15 M KCl solution were used, and the total charge of the system was adjusted by K+ and Cl− ions. The energy minimisation of the system was performed in 100,000 steps, using the steepest descent algorithm. The equilibration was performed with NPT ensemble and was maintained at 310 K using the V-rescale algorithm [38] and 1 atm under Berendsen barostat, as implemented in Gromacs 2016. 1. First run of equilibration was performed for R8 and R8-DLC1 with long linkers. These complexes were followed over 30 ns simulation without applied restrictions. Both domains were very stable, with low root-mean-square deviation (RMSD) changes. Major structural changes were observed for the long interdomain linker. In the second equilibration run, the fixed and pulled Cα atoms were restrained with harmonic potential during the 10 ns simulation. The temperature coupling was applied separately for the protein and the solution parts. Each system was equilibrated up to 10 ns, with subsequent monitoring of the RMSDs of Cα atoms. Structures after 10 ns were used as the starting pulling conformation. Pulling vector was set between Cα of the first and the last residue of the appropriate domain parallel to the z-axis of the pulling box. The movement of Cα of N-terminal residue was restrained with harmonic potential, while Cα of C-terminal residue was subjected to the constant velocity pulling. After placing the equilibrated protein in the pulling box and orienting it appropriately in the pulling direction, the water solution was equilibrated as external bath during 1 ns bath equilibration [39]. Furthermore, equilibration was performed at isotropic conditions while pulling at semi-isotropic conditions when pressure control was turned off in the pulling direction (z-axis). The constant velocity pulling SMD simulations were performed at 2 nm/ns with the spring constant set to 1,000 kJ/mol nm2. All trajectories were produced in 3 repetitions. The WT MEF cell line was a kind gift from Dr Wolfgang Ziegler and has been previously described by Xu and Baribault in 1998 [40]. The Tln1−/−Tln2−/− MEF cell line has been previously described by Theodosiou and colleagues [17]. Both cell lines were maintained in high-glucose DMEM supplemented with 10% FBS and 1% GlutaMax (Thermo Fisher Scientific, United States of America). A humidified 37°C incubator with 5% CO2 was used for culturing both cell lines. Cells were negative when tested for mycoplasma contamination. In all cell experiments other than the FRAP, a GFP-Talin1 plasmid was used. The GFP-Talin1 plasmid was a gift from Anna Huttenlocher (Addgene plasmid # 26724) [41]. The GFP-DLC1 plasmid was a gift from Irene Ng [42]. The GFP-vinculin plasmid was obtained from Susan Craig [43]. The GFP-paxillin was a gift from Chinten James Lim (Addgene plasmid # 80023). The talin 1 plasmid (Addgene # 26724) was used as template to generate the R8, C1, and C2 constructs by PCR-based mutagenesis starting construct via 5′ FseI and 3′ Scal. R8: For the substitution of amino acid glutamine with cysteine at position 1459, CAA was replaced with TGC; and for the substitution of serine for cysteine at position 1583, AGC was replaced with TGC. C1: For the substitution of amino acid glutamine with cysteine at position 1459, CAA was replaced with TGC. C2: For the substitution of serine for cysteine at position 1583, AGC was replaced with TGC. The GFP-DLC1 FRAP experiments were conducted on glass-bottom petri dishes (Mattek) coated with human plasma FN (10 μg ml−1; Sigma) and incubated at 37°C. For the DTT condition, DTT (Sigma) was added at a concentration of 100 μM 30 min prior to the start of the experiment. Confocal photobleaching and TIRF imaging were carried out using an inverted microscope (Eclipse Ti; Nikon). Five TIRF images were taken at 5 s intervals prior to bleaching for reference. Specified regions of the cells were then bleached using the confocal laser at 100% power. TIRF images were taken at 5 s intervals for 100 s to capture fluorescent recovery. Images were analysed with FIJI, with the fluorescent signal normalised between the prebleach intensity and background. Statistical analysis was then carried out using Prism (GraphPad). Data were pooled from repeats. The significance between curves was measured using extra sum-of-squares F test on the best fit lines. Immunofluorescence staining was done on coverslips coated with 10 μg/ml fibronectin (Sigma, F0895). Following pertinent treatment, cells were fixed with 4% PFA (Sigma, P6148) in PBS for 10 min and then blocked and permeabilised with 2% BSA-0. 1%Triton (Sigma, T8787) in PBS for 30 min. After blocking, cells were incubated with primary antibodies (MLC-2 Millipore MABT180 1/200 and pMLC-2 Thr18/Ser19 Cell Signalling 3674 1/200) prepared in blocking solution for 1 h at room temperature in a humidified chamber. Then cells were washed in PBS and incubated with Alexa Fluor 488 conjugated secondary antibodies and Phalloidin (Invitrogen, A22283,1/1,000 dilution) prepared in PBS for 30 min at room temperature. Finally, coverslips were washed in PBS and mounted in mounting reagent with DAPI (Invitrogen, P36931). GSH was purchased from Sigma-Aldrich (catalogue 78259) and used at a concentration of 10 mM. DLC1 antibody (clone C-12, catalogue sc-271915, dilution 1/100). siRNA for DLC1 was from Santa Cruz Biotechnology, catalogue sc-72134. Primers used to monitor DLC1 gene expression knockdown: DLC1 (m) -PR, catalogue sc-72134-PR. The micropillar arrays are based on our protocol as described previously [22]. Pillar arrays were coated with human plasma FN (10 μg ml−1; Sigma) and incubated at 37°C for 1 h before measurements. Cells that had been trypsinised before measurements were suspended in culture media and plated onto the pillar substrates. Time-lapse imaging of the pillars was conducted with an inverted microscope (Eclipse Ti; Nikon) operating in bright-field mode, with the samples held at an ambient temperature of 37°C. Image sequences were recorded with a sCMOS camera (Neo sCMOS Andor) at 0. 5 Hz using a ×40 (0. 6 NA, air; Nikon) objective over 100 min. The position of each pillar in the time-lapse videos was tracked using a custom MATLAB program to track the centre of a point spread function of the intensity of the pillars across all frames. By selecting a location free of cells, tracking of a small set of pillars allowed a measurement of the stage drift to be obtained and corrected for in the data set. The time-dependent displacement of a given pillar was obtained by subtracting the initial position of the pillar (zero force) from the position in a given frame. Traction forces were obtained by multiplying the pillar displacements by the pillar stiffness; the maxima for each pillar were found to obtain the peak forces across the cell. To analyse the ECM remodelling ability of MEFs, collagen-I (BD Biosciences, 354249, stock concentration 9. 37 mg/ml) and Matrigel (BD Biosciences, 354234, stock concentration 9 mg/ml) mixture gels were prepared with 10% 10× DMEM (Sigma, D2429) and 10% FBS (Gibco, 10500), yielding to a final concentration of 4. 4 mg/ml collagen-I and 2. 2 mg/ml Matrigel. The gel mixture was neutralised with 1 M NaOH (Sigma, S8045); then, 5 × 105 cells were embedded in gels in culture media. Eighty μl gel volume was added per well of a 96-well plate, which was pretreated with 2% BSA (Sigma, A8022) for 1 h, washed with PBS, and air dried for 10 min. Gels were set 1 h at 37°C and then incubated with culture media for 2 d at 37°C. Polystyrene 6-well plates were coated with 10 μg/ml fibronectin in PBS (pH 7. 4) at 37°C for 1 h and washed 2 times with PBS. Transfected Tln1−/−Tln2−/− MEF cells were allowed to recover for 24 h, trypsinised, and plated onto fibronectin-coated well plates at a low confluency. Cells were allowed to attach for 90 min, followed by replacement of media (2 ml of fresh media) to remove nontransfected cells. For treating the cells with diamide, diamide powder (Sigma-Aldrich D3648) was dissolved in PBS to a stock concentration of 50 mM (8. 6 mg/ml) and further diluted with complete cell culture media to 150 μM concentration. One ml of the diamide-containing media was added dropwise to 2 ml of cell culture media in each well 15 min before live cell imaging was started. EVOS FL Auto (Life Technologies, USA) equipped with a 20× objective and 37°C and 5% CO2 incubator was used for live cell imaging for 12 h at 120 s intervals. The resulting image stacks were analysed with ImageJ version 1. 50e with MTrackJ plugin [44,45]. For each plasmid construct or diamide treatment, 130–200 individual cells were traced from 3 fully independent experiments. Lysates of diamide-treated WT MEF cells were analysed by 2D SDS-PAGE to confirm the effect of diamide treatment on the level of cellular disulphide bonds. Cells at 80% confluency on 10 cm dishes were washed twice with warm PBS and treated for 120 min with 50 μM diamide dissolved in cell culture media. In negative controls, regular media were used instead. After the diamide treatment, cells were washed with ice-cold PBS and treated with 40 mM iodoacetamide in PBS for 5 min. Cells were lysed with 500 μl of RIPA buffer (50 mM Tris-HCl pH 7. 4,1% NP-40,0. 5% Na-deoxycholate, 0. 1% SDS, 150 mM NaCl, 50 mM NaF) supplemented with 40 mM iodoacetamide and Roche complete protease inhibitor cocktail. Cell lysates were incubated on ice for 30 min and centrifuged at 14,000 g for 20 min at 4°C to pellet cell debris. The concentrations of cleared lysates were determined with BCA assay and matched by diluting the lysates with RIPA buffer. For 2D SDS-PAGE, 120 μg samples of cell lysates were mixed with 2× Laemmli sample buffer without reducing agents, denatured at 95°C for 5 min, and loaded onto a medium-sized 1 mm thick 10%/4% polyacrylamide gel. The polyacrylamide gels were run at 25 mA current for 5 h, followed by vertical slicing of the gel with a clean scalpel to separate each lane. The gel slices were incubated in SDS-PAGE running buffer supplemented with 100 mM DTT for 20 min at room temperature to reduce disulphide bonds in the lysates. After the treatment, the gel slices were briefly washed with running buffer and treated with 100 mM iodoacetamide in SDS-PAGE running buffer for 10 min. The pieces of gel were placed horizontally on top of 1. 5 mm thick 10% polyacrylamide gels and ran at 10 mA current for 14 h. Proteins on the gel were visualised by using silver staining. All statistical analyses were conducted with the Prism graphical software (GraphPad, Software). Data were generated from multiple repeats of different biological experiments in order to obtain the mean values and standard errors (sem) displayed throughout. P-values have been obtained through t tests. Significance for the t tests was set at P < 0. 05, for which graphs show significance through symbols (*, P < 0. 05; **, P < 0. 01; ***, P < 0. 001; ****, P < 0. 0001).
Mechano-induced conformational changes and the unfolding of protein domains are cornerstones of mechanotransduction and regulate the interaction of proteins with other molecules. Talin is a prominent molecule in focal adhesions and one of the few proteins that simultaneously connects integrin receptors in the cell membrane with the actin cytoskeleton. This bridging position, owing to the cytoskeleton’s contractile nature, exposes talin to forces along its length. In this work, we studied the implications of the R8 domain unfolding in the downstream activity of deleted in liver cancer 1 (DLC1), which binds the talin R8 domain and negatively regulates Ras homolog family member A (RhoA). We created a talin mutant with the R8 domain resistant to mechanical unfolding and observed that cells expressing these talin mutants have altered patterns of focal adhesion dynamics and lower levels of actomyosin contraction. This leads to decreased traction forces and diminished cell migration. We propose a novel force-controlled molecular switch that refines the mechanism of talin-mediated focal adhesion activation, providing negative feedback during focal adhesion maturation. The broader effects of this talin-mediated mechanism need to be elucidated, as it might regulate multiple cellular events.
medicine and health sciences
chemical compounds
gene regulation
focal adhesions
carcinomas
cancers and neoplasms
green fluorescent protein
light microscopy
gastrointestinal tumors
organic compounds
liver diseases
fibroblasts
oncology
developmental biology
luminescent proteins
microscopy
gastroenterology and hepatology
connective tissue cells
molecular development
amino acids
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cysteine
small interfering rnas
animal cells
proteins
adhesion molecules
gene expression
connective tissue
chemistry
biological tissue
short reports
fluorescence recovery after photobleaching
hepatocellular carcinoma
sulfur containing amino acids
biochemistry
rna
organic chemistry
nucleic acids
cell biology
anatomy
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protein domains
biology and life sciences
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non-coding rna
Mechanotransduction in talin through the interaction of the R8 domain with DLC1