Mechanosensitive Piezo1 channels are essential mechanotransduction proteins in eukaryotes. Their curved transmembrane domains, called arms, create a convex membrane deformation, or footprint, which is predicted to flatten in response to increased membrane tension. Here, using a hyperbolic tangent model, we show that, due to the intrinsic bending rigidity of the membrane, the overlap of neighboring Piezo1 footprints produces a flattening of the Piezo1 footprints and arms. Multiple all-atom molecular dynamics simulations of Piezo1 further reveal that this tension-independent flattening is accompanied by gating motions that open an activation gate in the pore. This open state recapitulates experimentally obtained ionic selectivity, unitary conductance, and mutant phenotypes. Tracking ion permeation along the open pore reveals the presence of intracellular and extracellular fenestrations acting as cation-selective sites. Simulations also reveal multiple potential binding sites for phosphatidylinositol 4,5-bisphosphate. We propose that the overlap of Piezo channel footprints may act as a cooperative mechanism to regulate channel activity. Mechanosensitive Piezo1 channels transduce mechanical stimuli into electrochemical signals. Jiang et al. show that due to the bending rigidity of the membrane, the overlap of neighboring Piezo1 footprints produces a flattening of the Piezo1 footprints and a cation-selective conducting pore. This study suggests that the clustering of Piezo1 channels may tune its sensitivity to applied force. Patients carrying gain or loss of function Piezo mutations present various disease conditions, such as xerocytosis, arthrogryposis, loss of proprioception, and lymphedema2. Upregulation of Piezo2 activity correlates with inflammation-induced pain states3, whereas gain-of-function Piezo1 variants confer Malaria resistance in humans and animal models4.Piezos are homotrimeric mechanosensitive channels expressed in many eukaryotic cells. In vertebrates, two isoforms, Piezo1 and Piezo2, transduce various forms of mechanical stimuli into electrochemical signals that contribute to many biological functions, including somatovisceral sensation, proprioception, vascular development, blood pressure regulation, osmotic homeostasis, and epithelial growth8. Compared with the Piezo2 structure, the upper part of the Piezo1 pore is more dilated, rendering a funnel-shaped pore with a minimum radius at the lower constriction site. A previous simulation showed that this lower constriction site is too narrow to conduct ion and water9, indicating Piezo1 structures are nonconducting. In addition, the peripheral arms are arranged in spirals, giving Piezos a triskelion shape when viewed perpendicularly to the membrane plane and a bowl-like shape when viewed parallel to it. This bowl shape imposes a local convex curvature, or inverted dome, to the lipid bilayer, suggesting Piezo channels become flatter, and potentially open, when membrane tension increases11.Piezo channels sense mechanical cues transmitted directly from the membrane, and thus obey the so-called force-from-lipid paradigm. High-resolution cryo-electron microscopy (cryo-EM) structures of Piezo1 and Piezo2 reveal a unique molecular architecture consisting of three long peripheral transmembrane domains or arms, and a central region harboring a unique transmembrane pore and an extracellular (EC) cap domain14. Hence, the gating properties of Piezo channels may be modulated by the local channel density at the plasma membrane. Due to the intrinsic membrane bending rigidity, the membrane deformation induced by a single Piezo1 channel, or membrane footprint, is predicted to extend well beyond the local dome and decay within tens of nanometers10. Simulating Piezo1 channel in such a large membrane is feasible using coarse-grained (CG) molecular dynamics (MD) simulations, but remains challenging for all-atom (AA) MD simulations without sacrificing temporal sampling due to a large number of atoms. In most MD simulations with explicit solvent, the periodic boundary conditions (PBC) create an infinite lattice where the simulated system is infinitely replicated throughout virtual space. Thus, when Piezo1 is simulated in a membrane area smaller than its membrane footprint, the PBC creates a virtual cluster of channels with overlapping footprints between neighboring images.The formation of Piezo1 clusters has been reported by independent studies, which suggest Piezo1 clustering plays a role in concerted gating transitions, such as collective loss of inactivation16, we included 5% phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet of the 1-palmitoyl-2-oleoy phosphatidylcholine (POPC) bilayer to assess potential lipid–protein interaction during both CG and AA simulations. In good agreement with the hyperbolic tangent model, footprint overlap reduces membrane curvature, leading to a tension-free flattening of the Piezo1 arms and concomitant widening of the pore constriction site within 2 µs AA simulation. The conducting state generated from MD simulation reproduces ionic conductance and selectivity properties consistent with numerous experimental studies. In addition, we observed consistent PIP2 binding hotspots from multiple simulations and investigated the functional role of these putative binding sites, using functional assays.Here, using a hyperbolic tangent model, we first propose that the overlap of neighboring Piezo1 membrane footprints create membrane topology constraints that flatten the Piezo1 dome, facilitating Piezo1 opening. We tested this hypothesis using AA MD simulations of a crowded Piezo1 cluster with different degrees of footprint overlap. Given the importance of phosphoinositides to Piezo function14. The distribution of Piezo1–GFP puncta fluorescence intensity shows a main peak with secondary peaks at higher values, suggesting puncta correspond to clusters having a varying number of channels  for the hyperbolic tangent model , independent of the dome’s size. If we define the inclination angle α between the Piezo arm and the xy plane , the slope of the Piezo dome is tan−1(α).Using total internal reflection fluorescence (TIRF) microscopy, we found that GFP-tagged Piezo1 channels heterologously expressed in HEK293 cells exhibit a punctate distribution Fig. , consistels Fig. . Within del Fig. . Note R θ (larger θ means flatter footprint overlap) and the inclination of the Piezo1 arm  leads to a flattening of the Piezo dome and arms (decreased α). In contrast, when the dome angle is larger than this critical angle, the overlap flattening leads to more pronounced arm curvature (increased α). Thus, the hyperbolic tangent model suggests that when two Piezo footprints overlap, larger Piezo–Piezo distances favor arm curvature, while smaller Piezo–Piezo distances favor arm flattening. This qualitative biphasic relationship is independent of membrane mechanical properties. However, the exact value of this critical distance (separation coefficient D) depends on the geometric parameters of the footprint and bilayer rigidity. Below we show how this model corresponds to the parameters in MD simulations of Piezo1.This model predicts a biphasic relationship between the footprint overlap angle The hyperbolic tangent model suggests that the overlap of neighboring Piezo1 footprints favors flattening of the membrane at a sufficiently small Piezo dome–dome distance. We hence explore the influence of the footprint overlapping on Piezo conformation using AA MD simulations with PBC. The atomic model was built using a cryo-EM structure of mouse Piezo1 in a nonconducting state at 3.8 Å resolution (PDB ID 6B3R), solved in the detergent micelle in the absence of mechanical stimuli. Piezo1 channel is a large homotrimer with 38 predicted transmembrane helices (TM) per subunit. In this cryo-EM structure, TM1–12 are not present, and TM13–16 side chains are not fully solved. To avoid those structural uncertainties in the atomistic model, we only include TM17–38 for each subunit. Missing loops larger than 20 amino acids were not modeled (see “Methods” and “Discussion” sections). Although the arm length in this model is presumably 5/9 of the full length, our simulations show that this model encompasses key structural features, allowing Piezo1 to sense the membrane topology and open its pore.2 bilayer was first conducted to accelerate the convergence of lipid lateral diffusion  using an average centroid distance of a triangle defined by the center of mass of the outer helix in each arm  is hence the box dimension in x or y minus 2r. The smallest dome–dome distance in each simulation is 1–3 nm along the x-dimension and 4–7 nm along y-dimension  of the Piezo1 backbone plateaued after ~1 µs in all systems Fig. . By subtne Table . Becauseon Table . Hence, ges Fig. . Note thα from Piezo1 cryo-EM structure to be 38°. According to Fig. D) 1.6–2.8. If we borrow the height of the footprint (H) for a single Piezo1 from ref. R is estimated to be 16–28 nm  during the 1.75 µs simulation. This fitting approach revealed a reduction of membrane curvature in all three replicas Fig. . In the xz and yz plane, and plotted against box size along x-axis (x-dim) and y-dim individually  of one neighboring subunit. As a consequence, flattening motions in arms 1, 2, and 3 are likely to influence pore helix conformations in subunits 3, 1, and 2, , respectively  for 50 ns to avoid a sudden membrane tension drop due to lipid removal. The tension was then gradually reduced to zero during 112 ns. During the equilibrium, we found that only the largest pore in box1 system quickly allowed water and ions to diffuse through the pore and the hydrated pore was stable for the remainder of the simulation  and polarity of the pore lumen, hence is channel specific.We first applied 14.2 mN mion Fig. . In box2+ and Cl− ions transits across the upper and lower boundaries of the pore helix region is 39.7 pS with 85% confidence interval of 22.4–66.8 pS .To validate whether the water-conducting pore represents a realistic open state, we calculated the unitary ionic conductance using MD simulations. Constant electric fields corresponding to the transmembrane potentials of −250, −500, +250, and +500 mV were applied perpendicular to the membrane to all the atoms in the simulation box, in the presence of symmetrical 150 mM KCl concentration. The pore remains stable under all tested voltages Fig. . The ion pS Fig. . Alterna pS Fig. . Despiteity Fig. . The rep28. Using the stable open-state conformation, we computationally silenced the negative charge of E2133 to mimic the electrostatic effect of E2133Q mutant. After 5 ns equilibrium simulation, ion permeation was measured under 500 mV for 100 ns. This mutant simulation is designed based on the assumption that the conformational perturbation by the mutated residue is local, in other word, no a large and slow protein conformational change is needed to produce the mutant phenotype. As expected, this charge neutralization reduced the total number of permeation events under 500 mV from 18 events in WT to 8 events in E2133Q mimic mutant , E2383 in the cap, and D2006 located in the arm of a neighboring subunit. Several negatively charged residues are located along the narrower intracellular entryway, such as E2172 on the anchor, and E2487, E2495/6 on the inner helix  at the bottom of the pore remains closed. The three lower fenestrations observed from the ionic pathway start above this constriction neck. When the ions reach the bottom of the TM38 (E2495/6), they exit horizontally between neighboring base helices (L2149–Y2175). The radius profiles connecting the pore with lower fenestrations are shown in Fig. ed Table .2 lipids quickly diffuse toward Piezo1 within 1 μs and remain at the annular region of the protein throughout the 12 μs CG trajectory and the following AA simulations. A large number of PIP2 binding and unbinding events were observed from microseconds CG simulation and AA simulations, which allowed us to identify putative PIP2 binding hotspots in Piezo1. The binding distance is determined by the first minimum from the radial distribution function between arginine/lysine side chains and the PIP2 headgroups (see “Methods” section). Out of 143 cationic residues in each subunit of the current Piezo1 model, only 16 residues interact with at least one PIP2 over 60% of 12 μs CG simulation and over 90% of 2 μs AA simulation time  current amplitudes, while the K1727Q/R1728Q showed a 44% decrease at the maximal stimulation strength  on both the EC and intracellular sides. Lateral fenestrations are not uncommon as they have been identified in many families of ion channels and transporters. The multi-fenestrated permeation pathway of Piezo1 is also consistent with the observation that the deletion of a C-terminal beam-to-latch region of Piezo1, which forms a cytosolic plug under the central pore, does not yield constitutively open channels31. The intracellular lateral fenestration is also entirely consistent with the ion-conducting lateral portal reported recently30. In our MD simulation, K+ ions interact with acidic residues (mainly glutamate) known to contribute to ion selectivity both in the EC and intracellular fenestrations, as well as residues that have not yet been experimentally tested. We further validate this ionic pathway using simulated ionic conductance in WT, conductance-reducing mutant E2113Q in the anchor region, and a selectivity-reversing 9 K mutant in the lower fenestration.Simulating Piezo1 in a crowded environment induces global gating motions. Pore opening correlates with an outward motion of outer pore helices, allowing tilting of inner helices and rotation of V2476 side chains away from the pore lumen. In addition, we observed a concomitant rotation and a widening of pore-facing cap sub-domains. The rearrangements of pore-facing subregions of the cap have been recently shown as necessary for Piezo1 activation30. The latch region (1406–1420) under the CTD was partially solved in one of the cryo-EM structure (PDB 6BPZ). However, due to the large gap with the rest of the sequence, an additional constraint was necessary to keep this short loop in place during the simulation. Therefore, to avoid the uncertainty introduced by additional restraint, we did not include the latch in our simulation. Interestingly, the Piezo1.1 variant lacking the lateral plug domains exhibits three discrete sub-conductance levels, suggesting this lateral plug forms three additional activation gates, one for each of the three intracellular fenestrations. Since our Piezo1 model lacks the lateral plug, it is functionally more closely similar to the endogenous Piezo1.1 isoform identified in C2C12 cells30.Our Piezo1 model and available cryo-EM structures do not contain the disordered loop at the bottom of the Piezo1 from residues 1382–1405, which is termed central plug and lateral plugγ is the membrane tension, ΔA is the relative change in projected area, ΔGprotein is the free energy of protein conformational change in the absence of membrane tension, and ΔGmemb is the free energy cost of membrane deformations.In all available Piezo1 cryo-EM structures, only about two-thirds of each arm are resolved. Hence, the dome size in our current model is necessarily smaller than the dome of a full-length Piezo1. The free energy change associated with the Piezo1 opening transition can be approximated using Eq. , in whicG = ΔGprotein − ΔGmemb > 0), meaning the free energy cost of channel activation is larger than the free energy cost of membrane deformation. In our simulations, the Piezo1 footprint overlap provides additional free energy, ΔGoverlap, for deforming the membrane independently of tension:In single-channel condition , without tension, Piezo is in a nonconducting state (ΔG ≤ 0) thus indicates that ΔGmemb + ΔGoverlap ≥ ΔGprotein. To induce a 50% open probability without tension, the free energy contribution from the footprint overlap must be in a similar magnitude to the work needed to open a single channel under tension without footprint overlapping, which has been reported to range within 50–150 kBT (ref. 11).The tension-free opening  in our current model. In other words, under the same ΔGmemb and ΔGoverlap, a full-length Piezo1 model would be expected to open even more readily. It is perhaps not surprising that our first AA Piezo1 model with only 1/3 of each arm  didn’t generate an open state (the truncated model was aimed to enhance the sampling of spontaneous binding of Piezo1 agonist Yoda1)9. In this perspective, simulating the full-length arms resolved in the Piezo2 structure8 will be invaluable for investigating the role of these domains as mechanical levers.According to the classic Helfrich–Canham expression, the bending free energy of a homogeneous bilayer Δ13 measured the area distribution of Piezo1 puncta in cell membranes . These ranges of values are, on average, larger than the expected projected surface area of an isolated channel at rest, which is estimated to be ~500 nm2 for Piezo1 . Using patch-clamp electrophysiology, Gottlieb et al.14 observed that repeated mechanical stimulations eliminate inactivation of all channels present in the patch at once, rather than in a time-dependent manner as one would expect for a stochastic process affecting a population of independent channels. Although the mechanism underlying this collective loss of inactivation remains unclear, this result shows that local stress can affect many channels simultaneously, suggesting they are in close spatial proximity.Many independent studies suggest Piezo1 form clusters in cell membranes. Here, using TIRF microscopy, we show that the distribution of the brightness of Piezo1–GFP puncta has a long tail toward higher intensity, consistent with the presence of multiple channels per puncta Fig. . Using s11. The clustering effect produced by the PBC of MD simulations mimics a high-density channel cluster and imposes a flattening of the Piezo1-induced membrane footprints, reducing the curvature of the lipid dome and of the Piezo1 arms. This shows that Piezo1 gating is directly governed by membrane topology, which may be manipulated by various mechanical stimuli, as well as the spatial proximity between neighboring channels. This suggests that Piezo1 channels may positively cooperate to gate their pore.Our simulations underscore the exquisite interplay between local membrane geometry and Piezo1 curvature, a property highlighted from cryo-EM and atomic force microscopy studies33. Changes in bilayer thickness due to hydrophobic mismatch has been proposed to induce cooperative gating between neighboring MscL mechanosensitive channels and thus may contribute to cooperative gating of nearby Piezo channels. Other entropic contributions due to reduced membrane fluctuations in Piezo clusters may also collectively influence gating property. Whether and how those factors contribute together to the cooperative gating in Piezo clusters with different densities is of interest for further studies.Crowding-induced membrane footprint flattening may not be the only possible mechanism underlying concerted gating in clustered Piezo channels. Cooperative gating may also be governed by direct protein–protein interactions between nearby channels or by indirect interactions mediated by auxiliary proteinsIt should be noted that MD simulations using PBC aimed at studying Piezo1 clustering may lead to several caveats. First, due to the large size of molecular systems simulating Piezo channels and their large membrane footprints, it will be technically challenging to generate a microsecond-long trajectory of a low-density Piezo1 cluster, using available computing resources. Second, channels replicated under PBC condition are mirror images of each other. Thus, MD simulations under PBC cannot replicate spatial heterogeneity of channels in a real cluster. The predicted biphasic behavior of Piezo channels under different cluster densities may be better investigated using high-resolution biophysical approaches.2 binding hotspots is not surprising given the high density of positively charged residues located in the intracellular part of the protein. Most of the hotspots captured here are consistent with PIP2 binding sites reported elsewhere35. In addition, the flattening of the Piezo1 arms did not alter PIP2 density around those hotspots, suggesting PIP2 binding is independent of channel conformation. It has been shown that the presence of negatively charged PIP2 or phosphatidylserine in the inner leaflet promote channel activation36. In symmetric PC:PC bilayers, mechanical stress alone is sufficient to activate Piezo1, which indicates that PIP2 is not an absolute requirement for Piezo1 activation, but rather acts as a modulator37. Our mutagenesis data shows that individual neutralization of PIP2 binding hotspots had minor effects on mechanically induced channel activity, suggesting that a large number of PIP2–protein interactions might be necessary to collectively shift the thermodynamic equilibrium in favor of the open state.The presence of PIP2 with multiple binding sites on a large protein surface area is in sharp contrast to the binding of PIP2 to well-defined single binding sites in many PIP2-regulated ion channels, such as inwardly rectifying K+ channels38, TRPV5 channels39, and TRPM8 channels40. In TRPV5, the PIP2-bound structure shows an open conformation39, compared to PIP2-free structures, indicating that binding of a single PIP2 molecule per subunit is sufficient to open the channel. Regulation of Piezo1 by PIP2 through multiple binding sites is likely to be far more complex. Mutations of putative PIP2 interacting residues had no effect on the inactivation kinetics of MA Piezo1 currents. The quadruple Lysine K2182-85 also showed up on our simulations as a cluster frequently interacting with PIP2. This cluster is equivalent to residues K2166-69 in the human Piezo1 channel, deletion of which is associated with xerocytosis41 and displays markedly slower inactivation of MA Piezo1 currents. This may indicate that PIP2 binding to distinct sites in the channel has different effects on channel function. Since PIP2 depletion had no significant effect on Piezo1 inactivation15, it is also possible that the effect of those mutations on channel inactivation is independent of PIP2 binding. Further studies are needed to understand the complex effect of PIP2 on Piezo channel activity. It is also possible that PIP2 amplifies mechanical coupling, which may reduce the entropic cost associated with the conformational rearrangement of the arms, an effect explained by the population-shift theory of allostery43.The apparent interaction of PIP2 binding sites identified from multi-scale simulations suggests many lipid–protein interactions cooperate to shift the open/close equilibrium of the channel. This Piezo1 open state model thus provides an avenue for mechanistic investigation of disease mutations and small-molecule drug discovery.In summary, this study shows that the overlap of Piezo1 membrane footprints contributes to channel gating Fig. . The opehttps://github.com/wesleymsmith/MembraneFootprintInteractionModel/blob/master/MembraneFootprintIntersectionModel.ipynb). Firstly, the membrane approaches a flat plateau when distant from the Piezo dome . Lastly, if we place two domes a distance of D reduced units apart (note: D = R/H), their membrane footprints will intersect at a minimum distance of D/2 units apart. Putting this together, we attain the Eq. (h(x) is the height of the membrane above the top of the dome at a radial distance of x reduced units away from the edge of the dome, and m0 is the slope of the membrane at the edge of the dome tan−1(α). Correspondingly, we may calculate the slope of the membrane footprint under this model as in Eq. (h′(x) is the slope of the membrane footprint at position x. We may then calculate the slope of the membrane footprint at the point of intersection with another membrane footprint as in Eq. . To reduce computational time, we first conducted a 12 μs CG MD simulation of Piezo1 embedded in a solvated asymmetrical POPC membrane containing 5% PIP2 in the inner leaflet (Table z-axis and the bilayer dimension (xy) in CG simulation is 698 nm2. We assessed the equilibration of CG MD by monitoring PIP2 lipid density distribution P2 MARTINI model carrying −4 charge was parameterized to be consistent with the experimental data (see the section below). A 28.4 × 24.6 nm membrane bilayer was solvated with explicit water in a simulation box of 28.4 × 24.6 × 25.3 nm. A total of 150 mM NaCl was added to each simulation and kept the whole system charge neutral (hydrated potassium and sodium have the same particle type in CG model).The CG representation of Piezo1 was constructed from our previously AA mouse Piezo1 model based on the cryo-EM structure (PDB ID 6B3R), which includes residue 782–1365 (Piezo repeat C-F and beam), 1493–1578 (clasp), 1655–1807 (repeat B), and 1952–2546 2 in the plasma membrane is PIP2 with −4 charge, which indicates one of the phosphate groups being protonated47. The current PIP2 model in Martini force field (residue name POP2) is based on PIP2 with −5 charge. Hence, the charge on bead name P2 in Martini lipid POP2 is reduced from −2 to −1. Benchmark was done to compare the Martini CG PIP2 model with the AA PIP2 structure (residue name SAPI24) in the CHARMM36 lipid force field48. Since CHARMM SAPI24 has one more double bond than the Martini POP2 model, the bead name C3A is modified to D3A with its type changing from C3 to C4. PyCGTOOL was used to check the correct CG to AA mapping49. The new Martini PIP2 model (POP5) is provided in Tables 2 model between the CG and AA are listed in Tables https://github.com/reneejiang/cg_pip2_topology_files.The predominant form of PIP−1 nm−2, and an elastic network50 with a cutoff of 9 Å and a force constant of 500 kJ mol−1 nm−2. The CG simulation was executed in GROMACS (version 2016.4) simulation package with the standard Martini v2.2 simulation setting51. The protein and membrane system were built using a modified enhanced version of the INSANE (INSert membrane) CG building tool. All lipid models and parameters used in this study follow the MARTINI v2.0 lipids, with the addition of the modified Martini PIP2 model (POP5). The overall workflow of the simulations includes the initial construction of the Piezo1-embedded membrane, energy minimization, isothermal–isochoric (NVT) and isothermal–isobaric (NPT) equilibration runs, and NPT production runs. Briefly, each system was firstly energy minimized . NVT simulations were carried out for 0.5 ns at 310.15 K with a timestep of 10 fs. A timestep of 20 fs was used for the following NPT simulations. A cutoff of 1.1 nm was used for calculating both the short-range electrostatic and van der Waals interactions with the Potential-shift-Verlet algorithm applied to smoothly shifting beyond the cutoff. Long-range electrostatic interactions were calculated using the reaction-field algorithm implemented in GROMACS. The neighbor list was updated every 20 steps using a neighbor list cutoff equal to 1.1 nm for van der Waals. The temperature for each group  was kept constant using the velocity rescale coupling algorithm with 1 ps time constant. For the NPT equilibration step, semi-isotropic pressure coupling was applied using the Berendsen algorithm, with a pressure of 1 bar independently in the cross-section of the membrane and perpendicular to the membrane with the compressibility of 3.0 × 10−4 bar−1. The pressure in the newly built system was relaxed in a 30 ns simulation using the Berendsen barostat with a relaxation time constant equal to 5.0 ps. Three-dimensional PBC were used. The production step for each system ran for 12 µs using Parrinello–Rahman barostat with a relaxation time constant of 12.0 ps. At the end of each CG simulation, the protein and lipids were mapped into its atomistic representation using Martini backward mapping scheme. The reverse-mapped atomic structure was solvated with CHARMM TIP3P water and 150 mM KCl using the CHARMM36 force field52.CG simulation in the current study is designed to simply allow faster convergence of membrane topology, while keeping the secondary and tertiary structure of Piezo1 intact. Hence, the protein backbones were kept rigid using positional restraint with a force constant of 1000 kJ mol3 in xyz-dimensions, to reduce the AA system size to ~800,000 atoms. This AA system was first minimized using 50,000 steepest descent cycles in GROMACS, and then underwent six stages of equilibrium run at 310.15 K using AMBER18 CUDA package, as described in our previous Piezo1 simulation9. After equilibrium run on AMBER18, three replicas of systems were run on ANTON2 supercomputer with 2.0 fs timestep. Lennard-Jones interactions were truncated at 11–13 Å and long-range electrostatics were evaluated using the k-Gaussian Split Ewald method53. Pressure regulation was accomplished via the Martyna–Tobias–Klein barostat, to maintain 1 bar of pressure, with a tau (piston time constant) parameter of 0.0416667 ps and reference temperature of 310.15 K. The barostat period was set to the default value of 480 ps per timestep. Temperature control was accomplished via the Nosé–Hoover thermostat with the same tau parameter. The mts parameter was set to four timesteps for the barostat control and one timestep for the temperature control. The thermostat interval was set to the default value of 24 ps per timestep. All three replicas at the end of ~200 ns were cut into a smaller size  to take advantage of the high performance on Anton2 for longer production runs (Table −1 Å−1 was added between part of the beams (residue 1339–1365) and CTD (residue 2491–2546) to prevent the C-terminal of the beams drifting >30 Å away from the bottom of the pore. The amphiphilic helices (residue 1493–1553) were subjected to RMSD restraints for additional 90 ns at the beginning of Anton2 simulation to ensure the helical structures remain stable for the rest of microseconds production run.At the end of 12 μs CG MD simulations, the reverse-mapped AA system was truncated to a system with initial size of 21.6 × 22.5 × 16.1 nmns Table . In the https://github.com/wesleymsmith/Piezo_Membrane_Gauss_2D_Model). This gaussian distribution was modeled using a full covariance matrix formulation taking the form:C is the covariance matrix for the gaussian function, zo is the plateau height of the gaussian dome model, h is distance from the plateau to the peak of the dome model, and μ is the coordinates of the dome model peak. Since C is a covariance matrix, it is by definition positive semi-definite and symmetric. Therefore, for fitting purposes, this model was recast in terms of the standard deviations p1, p2, and angle of orientation θ such thatR is the rotation matrix corresponding to a rotation of θ radians in the xy plane. This has the added advantage that the two principal curvatures at the peak of the gaussian dome model can be given as h divided by the square of the standard deviation along the corresponding principal axis. The model was then fitted to the headgroup center of mass data for each frame using the python scipy.optimize library’s minimize function, using the default BFGS method.To estimate membrane curvature, the Piezo dome was modeled as a bivariate gaussian fitted to the center of mass coordinates of each leaflet’s lipid headgroups . The starting values of p1, p2, p1, p2, and their eigenvalues were used for θ was determined as the angle of the rotation matrix, which would transform h was forced to be negative to ensure a concave up dome. The results of this fitting were then used to plot curvature of each leaflet for each of the three membrane configurations  over a simulation period (τ), I–V curve gave the conductance of 39.7 pS (R2 = 0.93) with 85% confidence interval (CI 85) of 22.4–66.8 pS from Poisson distribution  with the standard deviation estimated from bootstrapping  and streptomycin . Cells were used up to 25–30 passages, when a new batch with low passage number was thawed. All cultured cells were kept in humidity-controlled tissue culture incubator with 5% CO2 at 37 °C. Cells were transiently transfected with cDNA encoding the mouse Piezo1 channel or its mutants tagged with GFP on its N-terminus in the pCDNA3 vector using the Effectene reagent (QIAGEN). Cells were then trypsinized and replated on poly-D-lysine-coated round coverslips 24 h after transfection. Whole-cell patch-clamp recordings were performed 36–72 h after transfection at room temperature (22°–24 °C), as described previously15. Briefly, patch pipettes were prepared from borosilicate glass capillaries (Sutter Instrument) using a P-97 pipette puller (Sutter instrument) and had a resistance of 4–7 MΩ. After forming gigaohm-resistance seals, the whole-cell configuration was established, and the MA currents were measured at a holding voltage of −60 mV, using an Axopatch 200B amplifier (Molecular Devices) and pClamp 10. Currents were filtered at 2 kHz using low-pass Bessel filter of the amplifier and digitized using a Digidata 1440 unit (Molecular Devices). All measurements were performed with EC solution containing 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH adjusted to 7.4 with NaOH). The patch pipette solution contained 140 mM K+ gluconate, 1 mM MgCl2, 0.25 mM GTP, 5 mM EGTA, and 10 mM HEPES (pH adjusted to 7.2 with KOH). Mechanical stimulation was performed using a heat-polished glass pipette , controlled by a piezo-electric crystal drive (Physik Instrumente) positioned at 60° to the surface of the cover glass, as previously described15. The probe was positioned so that 10-µm movement did not visibly contact the cell, but an 11.5-µm stimulus produced an observable membrane deflection. We applied an increasing series of mechanical steps from 12 µm in 0.5-µm increments every 5 s for a stimulus duration of 200 ms. The inactivation kinetics from MA currents were measured by fitting the MA current with an exponential decay function in pClamp, which measured the inactivation time constant (Tau). To calculate this time constant, we used the current evoked by the third stimulation after the threshold in the incrementally increasing step protocol in most experiments, except in cells where only the two largest stimuli evoked a current. In the latter case, we used the current evoked by the largest stimulus, provided it reached 40 pA.HEK293 cells were obtained from the American Type Culture Collection  and were cultured in minimal essential medium (Life Technologies) containing 10% (v/v) Hyclone characterized fetal bovine serum (Thermo Scientific), and penicillin (100 IU mlHEK293 cells were transfected with GFP-tagged Piezo1 and were plated on poly-D-lysine-coated 25 mm glass coverslips. Measurements were performed 36–48 h after transfection. Culture medium was replaced with the same EC solution used for electrophysiology and cells were mounted on an inverted Ti2E Nikon inverted motorized microscope. Excitation was provided by a 488 nm solid state laser through a 100× NA 1.49 TIRF objective; images were collected with an ORCA Fusion III camera using the Nikon Elements software, and were further analyzed in Image J.Electrophysiology data in Fig. Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1Reporting Summary


Type I interferons (IFNs) play an important role in the pathophysiology of systemic lupus erythematosus (SLE). While cross-sectional data suggest an association between IFN-induced gene expression and SLE disease activity, interest in this as a biomarker of flare has been tempered by a lack of fluctuation with disease activity in the majority of patients. This led us to question whether IFN-induced gene expression might instead be a biomarker of overall disease severity, with patients with high levels spending more time in an active disease state.Levels of five interferon-responsive genes were measured in the whole peripheral blood at baseline visit for 137 SLE patients subsequently followed for 5 years. Log transformed values were summed to yield a composite IFN5 score, and the correlation with various disease outcomes examined. Receiver operator characteristic analyses were performed for outcomes of interest. Kaplan-Meier curves were generated to compare the proportion of flare-free patients with high and low IFN5 scores over time.The baseline IFN5 score was positively correlated with the adjusted mean SLE disease activity index-2000, number of flares, adjusted mean prednisone dose, and number of new immunosuppressive medications over the subsequent 5 years. Optimal cut-offs for the IFN5 score were determined using Youden’s index and predicted more severe outcomes with 57–67% accuracy. A high baseline IFN5 level was associated with a significantly increased risk of subsequent flare.Measurement of the type I IFN signature is a useful tool for predicting the subsequent disease activity course. One of the most characteristic immunologic abnormalities associated with systemic lupus erythematosus (SLE) is elevation of type I interferons (IFNs) , 2. AlthDespite the lack of correlation between the IFN signature and disease activity in some previous studies, we previously showed that patients with high levels of IFN-induced gene expression were more likely to have had a recent flare of their disease and had a higher rate of subsequent flare . These oOne hundred and thirty-seven SLE patients satisfying 4 or more of the revised 1997 American College of Rheumatology (ACR) classification criteria for SLE  or 3 ACRDisease activity was determined using the SLE disease activity index-2000 (SLEDAI-2K) . Clinica2 normalized expression levels of 5 IFN-induced ubiquitously expressed genes  were then summed to generate a composite IFN5 score [For measurement of interferon (IFN)-induced gene expression, total RNA was isolated from the whole peripheral blood archived in PAXgene tubes (Applied Biosystems) and gene expression was quantified by NanoString using a custom array (nanoString Technologies), as previously described . Log2 noN5 score .U test was performed. The significance of the association between variables was determined using Spearman’s correlation coefficient. Kaplan-Meier curves were generated to plot the proportion of flare-free patients over time and the significance of the difference between curves determined using the Gehan-Breslow-Wilcoxon test. A receiver operating characteristic (ROC) analysis was performed to evaluate the ability of the IFN5 score to discriminate between various disease outcomes, and the optimal IFN5 scores for predicting each of these outcomes were determined using Youden’s index. All statistical analyses were performed using GraphPad software .For comparisons of differences between two groups, the Mann-Whiney 2 transformed normalized expression levels of 5 ubiquitously expressed IFN-induced genes (IFN5 score), as in our previous studies [ρ = 0.334, p < 0.0001) and a moderate negative correlation with age  and disease duration  [ρ = − 0.214 and 0.230, p = 0.012 and 0.007, respectively), the association with the SLEDAI-2K was not driven solely by these serologic components, since a positive correlation was also seen with the cSLEDAI-2K . When the clinical components of the SLEDAI-2K were examined separately, the IFN5 score was positively associated with the presences of arthritis  and mucocutaneous involvement . No association was seen between the IFN5 score and the presence of renal involvement (associations were not examined for descriptors with ≤ 10 patients), nor was there an association between treatment with anti-malarial or immunosuppressive drugs and IFN5 score.Demographic, clinical, and treatment information at the initial baseline visit for the 137 SLE patients are shown in Table  studies , 21. IFN 0.0043) , 22, 23.ρ = 0.626) and also with the adjusted mean corticosteroid dose (ρ = 0.720 for cAMS and ρ = 0.656 for flares) and the number of new additional immunosuppressive agents used (ρ = 0.531 for cAMS and ρ = 0.602 for flares) over the next 5 years, and both of these also correlated with the baseline IFN5 score . In contrast, baseline IFN5 scores were significantly lower in patients who were in LLDAS at least 50% of the time as compared to those who were not . Taken together, the data indicate an association between the IFN5 score and disease severity over the subsequent 5 years.Previous work suggests that patients who spend a substantial proportion of their time in LLDAS have better outcomes than those with more active disease , 24, 25.n = 16) had a 75% chance of being on a mean adjusted prednisone dose ≥ 7.5 mg and an 81.2% chance of being in a sustained high disease activity state with recurrent flares (median = 2) over the subsequent 5-year period. These findings indicate that the baseline IFN5 score can be used to predict with reasonable accuracy disease outcome over the subsequent 5 years.To further explore the association between disease severity and baseline IFN5 score, we examined whether baseline IFN5 scores could predict subsequent disease course. Outcomes examined included a persistent high disease activity state, as indicated by the failure to achieve LLDAS at least 50% of the time over the subsequent 5 years, and a requirement for sustained corticosteroid treatment, as indicated by a mean adjusted prednisone dose of > 6 mg. We also examined recurrent flares (≥ 2 subsequent flares during the 5-year follow-up) and the need for introduction of new immunosuppressive agents. Results of the ROC analysis can be seen in Fig. n = 87, 41.4% flare and 31.0% severe flare) or who were clinically inactive at the baseline visit . The average time to flare was 1.372 years for all flares and 1.470 for severe flares.Given the recently reported association between high levels of serum IFNα and risk of subsequent flare over the next year , we quesn = 22, mean = 47.41 ± 6.04). The demographic information for these two groups is shown in Table p = 0.044). Although there was a trend to a decreased time to flare in patients with high IFN5 scores, this only achieved statistical significance for severe flares in patients who were not flaring at baseline.To determine the impact of baseline IFN levels on the risk of subsequent flare, patients were stratified into low and high IFN5 scores based upon whether their IFN5 levels were > 2 SD above the mean of those for healthy controls (In this study, we show that several indicators of disease severity, including the mean clinical disease activity, number of flares, and amount of drug treatment, correlate with the baseline IFN5 score and that a single measurement of this score with appropriate cut-offs can be used to predict with moderate accuracy a more severe disease outcome over the subsequent 5 years. We further show that an elevated baseline IFN5 score is associated with a significantly increased risk of subsequent flare. While it could be argued that this association results from the observation that patients with high IFN5 scores have a higher baseline SLEDAI and that these patients are more likely to have subsequent flares, the same association was seen between IFN5 score and risk of flare in patients who were not flaring or inactive at baseline, suggesting that having a high IFN5 score confers an increased risk of flare independent of baseline disease activity.Although the IFN5 score was associated with indicators of more severe inflammation, no association with increased organ damage was seen. It is currently unclear whether the 5-year follow-up period was too short for significant damage to develop or whether other genetic and environmental factors play a more important role than IFN in determining the extent of damage that occurs in response to inflammation.The genes that comprise the IFN5 score are all found within a cluster of genes that has previously been shown to be strongly induced by IFN-α , 9, and In SLE, immune complexes containing RNA or DNA have been shown to promote IFN-α production by plasmacytoid dendritic cells through activation of toll-like receptors 7 or 9, respectively , 27. ThuThe concept that the levels of type I IFN may define overall disease severity rather than contemporaneous flare is supported by studies with type I IFN-targeted therapies. In a recent trial of anifrolumab, an anti-IFN-α receptor antibody that blocks the activity of IFN-α, IFN-β, and IFN-ω, approximately twice as many SLE patients achieved a low disease activity state at week 52 following initiation of treatment as compared to placebo control . NotablyA potential limitation of our study is that we examined an unselected prevalent SLE patient cohort of convenience with varying ages and durations of disease. It is possible that at disease inception the levels of IFN-induced gene expression may not be as informative. Studies show that following disease onset patients can follow one of three disease courses, persistent disease activity, recurrent remission and exacerbation, and initial disease activity followed by prolonged disease quiescence . WhetherMeasurement of the type I IFN signature at a single time point may be a useful clinical tool for predicting the subsequent disease activity course, with a low IFN5 score bolstering the clinician’s confidence that the patient is likely to have a less active disease course and decreased risk of subsequent flare. In contrast, clinicians should be wary of patients who have very high IFN scores given the high likelihood of more severe disease and may wish to consider more aggressive treatment in this subset of patients.


To assess color compatibility between dental structures (human enamel and dentine) and three different types of ceramic systems.00/ΔE*ab) and corresponding Coverage Error (CE) of ceramic system for dentine or enamel samples were calculated. Color data was analyzed using one-way ANOVA and post-hoc multiple comparisons tests. CE values were interpreted by comparisons with available 50:50% acceptability color threshold (AT) for dentistry.Samples (1 and 2 mm-thick) of extracted tooth (containing dentine and enamel areas) and three ceramic systems with different shades and opacities  were prepared for this study: Vita Suprinity—VS  (Vita Zahnfabrik); Vita Enamic—VE  (Vita Zahnfabrik) and Noritake Super Porcelain EX-3—NKT  . Reflectance measurements of all samples were performed over black backgrounds using a non-contact spectroradiometer  under a CIE 45°/0° geometry. CIE L*a*b* color parameters were measured and CIELAB/CIEDE2000 color differences  and for 2 mm-thick  enamel samples.Statistically significant differences in lightness were found among all ceramic systems and human dentine (p < 0.001), while no significant differences were registered between enamel and VSHT, T and VEHT. 1 mm dentine showed no statistical differences with VST and VSHT for a* coordinate, while 2 mm dentine showed no significant differences (p > 0.05) with VEHT. Thin samples (1 mm) of dentine and enamel showed significant statistical differences (p < 0.05) for b* coordinate with less translucent materials . For dentine samples, none of the ceramic materials provided a CE lower than AT. VSHT provided the best CE for 1 mm-thick (CE2 lithium silicate glass–ceramic (VS), with its two levels of translucency, provided lower CE values with human enamel samples while conventional feldspathic ceramic (NKT) and hybrid ceramic systems (VE) demonstrated a better color compatibility with dentin samples.Color coordinates of evaluated esthetic ceramic systems were statistically different from those of human dentine in almost all cases. The evaluated ZrO The wide range of available all-ceramic systems, with ever improving mechanical and optical properties, are trying to reproduce, to a great extent, the properties of natural dental structures , 2.The complexity of tooth color, however, seems difficult to achieve by any existing restorative material. Light reaching tooth surface is partly absorbed, diffused, transmitted or reflected by hard dental structures. Recent research revealed that these physical optical phenomena, occurring at the surface and inside tooth structures, are strongly influenced by the tooth type . NowadayConventional feldspathic ceramic is considered to be the most translucent ceramic material, the “gold standard” in esthetic dentistry, but on the other hand is brittle and has a very low resistance to fracture , 6. SuccComputer-aided design and computer-aided manufacturing techniques (CAD/CAM) have gained ground over the years. Using these procedures, several materials with large clinical indications, including zirconia, alumina, lithium silicate and disilicate based ceramics can be fast and easy milled –11. HybrZirconia-reinforced lithium silicate glass–ceramic, containing more than 10% Zr has been recently introduced for aesthetic reconstruction of posterior teeth. This CAD/CAM processed material proved to be strong, fracture resistant and natural looking, having similar optical properties [However, there is a limited number of studies that report on the color compatibility of these ceramics with the hard tissues they are supposed to replace. Therefore, it is essential to further investigate the optical and colorimetric properties of these materials, in comparison with the enamel and dentine, in an effort to understand their behavior and to provide a scientific support for clinical decision-making.. The optical color parameters as well as the translucency depend on the type of material and its thickness [In esthetic dentistry, the choice of the most appriopiate material of restoration represent the keys to the succes of the final clinical result . The opthickness , 17. In nt}ΔE00) , 19. In nt}ΔE00) , 22.The use of color differences in dental research is wide, covering diverse areas such as shade correspondence , instrum2 lithium silicate machinable ceramic, hybrid ceramic and feldspathic ceramic). The null hypotheses tested were: (1) Color parameters of analysed ceramics did not differ from the corresponding values of human enamel and dentine; (2) the CE for enamel and dentine of all studied materials is below or at the clinically acceptable threshold for color in dentistry.The main objective of this study was to test the color compatibility between dental structures (enamel and dentine) and three different types of ceramic systems, considered as representative for their class . Zirconia reinforced lithium silicate glass–ceramic samples were sliced in their precrystallized condition. The slices were then submitted to a crystallization process in a furnace , according to the manufacturers’ instructions. The conventional feldspathic ceramic samples were fabricated with a porcelain sampler . This system facilitated the manufacturing of very precise round samples of a determined thickness  increasingly with wet 120-, 240-, 400-, 600-, 800-, and 1200-grit silicon carbide paper. The final thicknesses of all samples were established to 1 (± 0.2) mm and 2 (± 0.2) mm as determined with a digital micrometer  with an accuracy of ± 0.05 mm.Freshly extracted human posterior teeth obtained from a tissue bank were preserved in sodium azide solution  prior to sectioning. Before cutting them, they were visually inspected and cleaned from debris and only those that were free of caries, restorations or any other pathological discolorations were included in the study. A low speed diamond saw  was used to cut approximately 1.2 and 2.2 mm-thick sagittal bucco-lingual tooth slices, each containing both enamel and dentine substrate  with distilled water for 10 min and dried under compressed air before they were measured.Three short-term repeated reflectance measurements in the 380–780 nm range  were performed for each sample inside a completely dark room using a spectroradiometer , a fiber-coupled Xe-Arc light source  and a Spectrally Calibrated Reflectance Standard . Between the samples and the black background, a saturated sucrose solution (refractive index n = 1.5 approximately) was interposed. The spectroradiometer was placed 40 cm away from the samples and the illuminating/measuring geometry corresponded to CIE 45°/0°  were calculated using both the CIELAB  was calculated as the mean value of the minimal color differences  and 50:50% Acceptability Thresholds—AT—, as reported in a multi-center study [CE values were compared with clinically relevant 50:50% Perceptibility Thresholds—PT— of these variables have been computed for all investigated enamel, dentine and ceramic samples. Confidence intervals for mean CIE L*,a*, and b* have also been bootstrapped based on replications of 1000 samples. The normality of the measured color parameters has been investigated using Kolmogorov–Smirnov, Shapiro–Wilk tests and Quantile–Quantile Plots and the homogeneity of variance was assessed by Levene’s test. One-way ANOVA was used to compare the mean values of L*,a*, and b* color coordinates for all ceramic and tooth samples of different opacities, shades and thicknesses. Multiple comparisons using Tamhane’s error correction procedure have been performed for post-hoc comparisons of the measured color parameters between enamel, dentine and ceramic samples. The level of statistical significance has been set at α = 0.05 .Means, standard deviations and results of statistical analysis of CIE L*a*b* color coordinates of the evaluated materials for the two analysed thicknesses are presented in Table p < 0.001). In the case of enamel, for a thickness of 1 mm, the most similar values of mean lightness were encountered for VSHT and VST , while for a thickness of 2 mm, VSHT, VST and VEHT showed no significant differences with enamel samples of the same thickness . Besides, when the 2 thicknesses of the same tooth structure were compared with each other, no significant differences in lightness were found between enamel samples of 1 and 2 mm (p = 1.000) and between dentine samples of 1 and 2 mm thickness (p = 0.966).The results of one-way ANOVA based on ranked data showed highly significant differences in CIE L* mean values among dentine samples of 1 or 2 mm thickness and all the other materials, including enamel (p < 0.001), excepted for VSHT (p = 0.977) and VST (p = 1.000) when compared to dentine. The correlations between dental structures and ceramic materials for CIE a* values increased with the thickness of the material, showing no significant differences (p > 0.05) between dental structures of 2 mm thickness and the 5 materials measured, except for human dentine versus NKT, VET, VST and VSHT, and human enamel versus VET .Highly significant differences were identified between the mean CIE a* values of 1 mm-thick dentine and enamel samples and the other materials tested (p = 1.000). For thin samples (1 mm), both dentine and enamel samples showed significant statistical differences (p < 0.05) only with the less translucent materials tested . No statistically significant differences (p > 0.05) were found for the CIE b* chromatic coordinate between human enamel and 2 mm-thick dentine samples and all three ceramic systems studied.CIE b* values of dentine samples revealed very close mean values to enamel samples of the same thickness  of the different ceramic systems studied for color of dentine or enamel samples (1 and 2 mm) are represented in Figs. 00 = 5.8; CEab = 8.4), while for 1 mm dentine samples, the lowest CE was found for the VET system . In all cases and for both color difference formula used, the CE values found exceeded both PT and AT. In the case of 2 mm thick enamel samples, the best CE was exhibited by the VST ceramic system (CE00 = 2.2) and the VSHT ceramic system (CEab = 2.5), depending on the color difference formula used for computation. For 1 mm-thick enamel samples, the VSHT systems showed the lowest CE . The VS systems, in it´s both translucencies (VSHT and VST), showed CE values higher than PT but lower or at AT, independently of the thickness of the sample  were thoroughly analysed from a colorimetric point of view.Nowadays, the increasing number of available all-ceramic materials leads to difficulties in choosing the proper material for the desired dental restoration. Aesthetic properties, along with composition and mechanical properties of restorative materials represent the main factors with an impact on the clinically optimal material selection. In the present study, the color coordinates of three types of all-ceramic materials with different opacities and different shades were comparatively evaluated with samples of human dentine and enamel of corresponding thicknesses. Being able to relate the properties of dental restoratives with human dental hard structures is of great importance , since tSpectroradiometric objective color measurements are the current standard in dental research –35, sinc2SO3) and lithium orthophospates (Li3PO4) are present, led to an increased translucency when compared to the two others ceramic systems [2O3 from its composition and to the crystal size (up to ~ 20 μm) and structure, which does not resemble to any particular mineral [The first null hypothesis of this study was partially rejected, as statistically significant differences in lightness between dentine samples and all the studied materials were registered, while for enamel, several materials  showed no significant differences in CIE L* mean values when compared to 1, respectively 2 mm-thick samples. It is well known that lightness is strongly correlated to translucency . A trans systems . Besides mineral , 38. AccIt was reported that the reddish and yellowish appearance of ceramics depends on its thickness , similarAlthough 1 and 2 mm-thick specimens are both clinically relevant as well as frequently used in dental research when studying color and optical properties of tooth structures (enamel or dentin) and dental materials  , 41–43, 00 = 0.8 or ΔE*ab = 1.2 and 50:50% AT of ΔE00 = 1.8 or ΔE*ab = 2.7, as reported in a multi-center study [Although CE is used in dentistry –29 mainler study . A realiNone of the ceramic materials included in this study provided a CE lower than the corresponding AT for the dentine samples. However, CEs lower or at the AT threshold were found for VSHT and VST for enamel samples of both thicknesses Fig. . AccordiIn all cases, for corresponding thickness of dentine and enamel, all ceramic systems analysed consistently provided a better CE for enamel samples than for dentine samples, as expected, since the background used for objective color measurements was black and enamel samples are highly translucent. Therefore, the second null hypothesis of this study is also partially rejected, since the CE for enamel and dentine samples of all studied materials is higher (with few exceptions) than the clinically acceptable threshold for color in dentistry. It would be of real interest to confirm these results by comparing these different types of ceramic materials with a larger number of enamel and dentine samples of different thicknesses, obtained from different types of teeth with a large range of color. Furthermore, it cannot be disregarded that natural enamel and dentin are mainly anisotropic materials while the dental ceramics are isotropic materials. This implies that the direction of the incident light or the analysis of different parts of enamel and dentin may lead to different optical properties.2 lithium silicate glass–ceramic in its both translucency version (T and HT) could be a proper choice for veneers and crowns where only enamel and a small amount of dentine has to be replaced. Similar to previous studies [From a clinical point of view and taking into account the results of the present research, machinable ZrO studies , Vita En2 lithium silicate glass–ceramic (VITA Suprinity) with its 2 levels of translucency, provided lower CE values (better color compatibility) with human enamel samples.Color coordinates of evaluated esthetic ceramic systems were different from human dentine in almost all cases. The evaluated ZrONoritake Super Porcelain EX-3 conventional feldspathic ceramic and Vita Enamic hybrid ceramic systems were more color compatible with human dentine samples, although the CE of both ceramic systems exceeded the 50:50% acceptability threshold for both dentine thicknesses studied.


Schizosaccharomyces pombe, and provides prospect for future comparative studies.To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases, such as cancer or developmental disorders. Here, we compared mitotic and meiotic spindles in fission yeast. We show that, even though mitotic and meiotic spindles underwent the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of the kinesin-14 family protein Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of the kinesin-5 family protein Cut7 remains constant. We identified the second kinesin-14 family protein Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines the differences between mitotic and meiotic spindles in fission yeast This article has an associated First Person interview with the first author of the paper. Highlighted Article: Chromosome segregation requires the spindle apparatus in both mitosis and meiosis. We report that kinesin-14 family proteins and microtubule dynamics function differently in mitosis and meiosis. Cell division is an essential feature of all living organisms. Mitosis allows cell renewal and amplification by producing two daughter cells of the same ploidy as the mother cell. Meiosis promotes genetic mixing within species by producing four gametes of halved ploidy . In bothSchizosaccharomyces pombe is a simple, genetically tractable organism with many of its genes conserved in humans. Importantly, fission yeast can complete mitosis and meiosis in the same environment. This allows robust simultaneous study of both processes and provides an opportunity to understand the intrinsic differences in their spindle dynamics.As it is well recognized that irregularities in spindle dynamics can result in chromosome segregation errors, potentially leading to cancer, congenital diseases and cell death , knowingIn fission yeast, the initial assembly of the spindle occurs in phase I, during prophase/prometaphase, when nucleation of MTs from the spindle pole body  starts inside the nucleus A. MTs arHaploid fission yeast cells can undergo mitosis or, in nutrient-poor conditions, cells of opposite mating type can fuse and form a zygote B. A zygoIn most organisms, including fission yeast, deficiency of kinesin-5 family proteins leads to monopolar spindles during mitosis and failure to segregate the chromosomes . It has wt) fission yeast cells expressing the α-tubulin Atb2 tagged with mCherry (mCherry-Atb2) and the SPB component Sid4 tagged with green fluorescent protein (Sid4-GFP), cultured in malt-extract (ME) medium to induce mating. Fission yeast MTs reorganized at the end of vegetative interphase (IP) to build a mitotic spindle, with typical three-phase dynamics . We found that they are similar, except that the spindle elongation velocity in phase III is slightly slower in cells undergoing mitosis in ME medium, which results in ∼10% longer duration of mitosis . In zygotes , consistent with a previous report (wt and cut7Δpkl1Δ cells expressing GFP-Atb2 and Hht1-mCherry (histone H3) to visualize chromosome segregation. Although the mitotic spindle in cut7Δpkl1Δ cells was capable of segregating the DNA into two separate pools of equal size : 74±2% of cut7Δpkl1Δ zygotes failed to establish a bipolar spindle and separate the SPBs during MI, consistent with a previous report . An increase in SPB number after MI spindle failure could indicate that SPBs in cut7Δpkl1Δ zygotes were duplicated twice, and that cut7Δpkl1Δ zygotes entered MII despite failure of chromosome segregation in MI. We confirmed this result by imaging wt and cut7Δpkl1Δ zygotes expressing Cdc13  tagged to GFP (Cdc13-GFP) and mCherry-Atb2. In wt zygotes, Cdc13-GFP localization was visible in the nucleus, on the spindle and SPBs . The pool of Cdc13-GFP was replenished upon MII phase I onset, and was degraded again upon phase III onset of MII. Similar dynamics of Cdc13-GFP were visible in cut7Δpkl1Δ zygotes that formed monopolar spindles, except that Cdc13-GFP degradation relative to the onset of spindle assembly was delayed by 16 min on average in cut7Δpkl1Δ MI . This suggests that the spindle assembly checkpoint (SAC) is active but leaky in cut7Δpkl1Δ zygotes, ultimately allowing progression through the meiotic stages regardless of spindle or chromosome segregation defects.In s in MII D. In theand SPBs . Upon onwt and cut7Δpkl1Δ spindle dynamics. In mitosis, pre-phase III spindles were shorter and phase III onset was delayed in the cut7Δpkl1Δ mutant compared to that in wt . This phenotype is similar to the phenotype of mitotic cells that lack Cut7  tagged to GFP  . The anaack Cut7 .We conclude that deletion of Pkl1 cannot fully compensate for the absence of Cut7 in MI. This suggests that Cut7 and Pkl1 are differently regulated between mitosis and MI.wt mitosis and MI. We first checked the impact of the GFP tag on motor functionality. Cut7 deficiency is lethal in fission yeast , but no difference in growth was observed in serial dilution plate assay . Mitotic and MI spindle dynamics of the strain with the Cut7-GFP were comparable to the strain not expressing Cut7-GFP . These results indicate that the GFP-tagged version of Cut7 was mostly functional.To assess whether Cut7 and Pkl1 are, indeed, differently regulated in mitosis and MI, we imaged Cut7 or Pkl1 tagged to GFP together with MTs (mCherry-Atb2) in on yeast , resultiFig. S3A; 94% compared to 97% for the strain without GFP-tagged Pkl1) or any consequence on the growth in the serial dilution plate assay . Mitotic spindle dynamics of the strain with Pkl1-3×GFP were similar to those of the strain without GFP-tagged Pkl1 , whereas MI spindle dynamics showed a longer phase II duration and a shorter phase II spindle in MI . The results indicate that Pkl1-3×GFP is mostly functional.Pkl1 deficiency has been reported to lead to protrusions in phases II and III in 85% of the mitotic cells grown in YE5S medium . We obseDespite slight changes regarding spindle dynamics, as well as the observed protrusions and decreased percentage of zygotes with four spores that suggested a partial but not complete loss of functionality, we observed no changes in phase I. Therefore, we concluded that Cut7-GFP and Pkl1-3×GFP are suitable to investigate localization and recruitment of Cut7 and Pkl1 during phase I in both mitosis and MI.Similar to previous observations by We then measured Cut7-GFP and Pkl1-3×GFP signal distribution along the spindles during phase I to phase II transition by line scan analysis, 1 min before the spindle reached a constant length that marks the beginning of phase II.Fig. S3G,H). Because the total MT intensity is different in mitotic and MI spindles , we normalized the intensity values obtained for Cut7-GFP and Pkl1-3×GFP to the MT intensity values. This way we found that the relative concentration of Cut7 remained unchanged . We next compared mitotic and MI spindle dynamics in the triple mutant. MI spindle dynamics comparison revealed that, not only is spindle bipolarity re-established but the usual three-phase spindle elongation dynamics are partially rescued . This result was unlike the one noticed in cut7Δpkl1Δklp2Δ MI spindles, whose pre-phase III spindle length was partially restored  and showed a non-significant decrease in the percentage of zygotes producing four spores . The velocity of spindle elongation in phase III of mitotic spindle dynamics was faster and phase II duration was increased in the strain with tagged Klp2 . Tagging Klp2 resulted in a longer phase II spindle in MI  and two of the 17 observed zygotes required more time to reach spindle bipolarity. These results indicate that Klp2-GFP is mostly functional.Klp2 is a minus-end directed motor that can crosslink and slide MTs, thereby affecting spindle length . We taggImaging of Klp2-GFP showed that it localized along the spindle, starting from spindle assembly until early phase III in both mitosis and MI  Fig. 4DDFig. 4D.klp2 deletion rescued spindle bipolarity only partially in the cut7Δpkl1Δ zygotes, we searched for additional factors that participate in force-balance in MI phase II spindles. We considered that MT dynamics might be differently regulated between mitosis and meiosis. Current imaging limitations prevented us from measuring individual MT dynamics within the spindle, where there are many crosslinked MTs; instead, we measured individual MT bundle dynamics before onset of mitosis (interphase), and in the pause period between meiotic MI and MII (interkinesis). We compared the dynamics of individual MT bundles that grew straight and originated from SPBs. Parameters defining MT dynamics during interphase and interkinesis are provided in Considering that cut7Δpkl1Δ zygotes.Kymographs and plots of MT bundle dynamics in a vegetative cell, and a zygote are shown in wt zygotes on ME plates with 5 µg ml−1 MBC resulted in a slightly decreased frequency of zygotes with four spores (cut7Δpkl1Δ zygotes produced four spores more frequently with the addition of MBC (44±1% in the presence of MBC compared to 20±1% without MBC). Live-cell imaging showed that addition of 5 µg ml−1 MBC allowed bipolar spindle assembly in MI within 80±4% of cut7Δpkl1Δ zygotes compared to 26±2% of cut7Δpkl1Δ zygotes when no MBC was added .We perturbed MT dynamics by using low doses of methyl benzimidazole carbamate (MBC), a drug that inhibits MT polymerization and suppresses MT dynamics . Mating r spores B. In conwt zygotes treated with MBC, the MI phase II spindle frequently exhibited transient breakage . The relationship between wt and cut7Δpkl1Δ spindle dynamics with the addition of 5 µg ml−1 MBC appeared similar to their relationship without MBC addition , were higher in MI than in mitosis. In fission yeast, spindle length scales with cell size . A simplWe further found that in MI, unlike mitosis, the largest proportion of time was spent in phase II . In MI, Finally, zygotes spent a similar time in phase I of the two meiotic divisions , but theFig. S5C). We have also considered that the kinetochores might not be attached to the spindle MTs in cut7Δpkl1Δ MI. Because of the bouquet rearrangement, the kinetochores are far from the SPBs during MI, making it relatively easy to detect kinetochore attachment to MTs. Imaging the kinetochore component Mis12 tagged to GFP and the SPB component Sid4 tagged to mCherry did not reveal a defect in kinetochore attachment .It has been reported that failure to form a meiotic bouquet leads to aberrant spindles . Howevercut7Δpkl1Δ background. Individual deletion of the meiosis-specific cohesin Rec8, the meiotic kinetochore protein Moa1, the meiotic sister-chromatid cohesin Rec11 or the meiotic recombination endonuclease Rec12 did neither rescue the cut7Δpkl1Δ MI spindle phenotype nor the number of zygotes producing the typical four spores . Interestingly, a recent study did report that the double-deletion of Rec12 and Moa1 partially rescued spindle bipolarity in a cut7Δpkl1Δ background . Nevertheless, by measuring the fluorescence intensities in the same cell but during different stages of cell division, we argue that the differences we observed were, indeed, intrinsic to the type of cell division, and not artifacts due to the fluorescent tag. It has recently been published that, in mouse oocytes, weak overexpression of the kinesin-14 family protein HSET  during MI accelerates spindle bipolarization during phase I, and results in more focused spindle poles, essentially switching from MI to a more mitotic-like spindle . These transient dynamic MTs could be newly polymerizing MTs or existing pivoting MTs. The fact that suppression of MT dynamics by addition of MBC rescued spindle bipolarity in cut7Δpkl1Δ zygotes, suggests that MT dynamics also contribute to the force-balance establishment within meiotic spindles, possibly by limiting the outward pushing forces generated by MT growth.In vegetative cells, centromeres are grouped at the nuclear envelope, below the SPB . In zygoOur results open new questions on how motors, MAPs and MT dynamics are modified during mitosis and meiosis. Given the high degree of evolutionarily conserved molecular players involved in spindle assembly, it will be most interesting to expand this study further towards other organisms, to develop our global understanding of mitosis and meiosis.S. pombe wild-type 972 strain, and were obtained using genetic crosses, selected by random spore germination and replicated on plates in the presence with appropriate drugs or supplements. All strains are listed in Table S1. Gene deletion and tagging was performed within the endogenous locus, except for Sid4-GFP and Mis12-GFP, which were integrated into the leu1-32 locus as described previously (600nm=0.5–0.8) were harvested by centrifugation and washed with 10 mM Tris HCl pH 7.4. Cells were centrifuged again and re-suspended in 100 mM lithium acetate supplemented with 10 mM DTT. The suspension was then incubated on an orbital wheel at room temperature for 40 min. Thereafter, 100 µl of suspension was mixed with 80 µl of 100 mM lithium acetate, 10 µl of single-stranded DNA from salmon testes  and 1 µg of the purified DNA. After 10 min of incubation on an orbital wheel, 300 µl of PEG 4000, dissolved in 100 mM lithium acetate was added. After incubation of 10 min on the wheel, 15 µl of DMSO was added. The mixture was then heat shocked at 42°C for 20 min. Cells were washed with distilled water and plated onto plates containing yeast extract medium supplemented with Leu, Ura, Ade, His, and Lys (YE5S). After 2 d of incubation at 25°C, the plates were replicated on selection plates containing the corresponding selective medium.All strains used in this work are isogenic to the eviously . TransfoS. pombe strains were maintained at 25°C and cultured in standard media. Generally, two days before imaging, cells were refreshed on a plate containing YE5S. The following day, cells of opposite mating types were mixed on an ME plate, or an ME plate supplemented with 5 µg ml−1 MBC, to induce conjugation. The plate with mating cells was incubated at 25°C for 16–20 h before imaging. For spore quantification, the mixture was incubated on an ME plate, or an ME plate supplemented with 5 µg ml−1 MBC, at 25°C for two days and subsequently imaged.All 600nm was 1.5. This suspension was serially fourfold diluted, 5 µl of the prepared dilutions was inoculated on plates containing YE5S and then incubated at 25°C or 30°C for 48 or 96 h.Serial dilutions were performed by growing tested strains on a plate containing YE5S at 25°C for 24 h, and then resuspending some of the patch in water so the OD−1 MBC. Imaging was performed at 25°C. Images were acquired on an inverted Spinning Disk Confocal (Roper/Nikon), equipped with Plan Apochromat 100×/1.4 NA objective lens (Nikon), a PIFOC (perfect image focus) objective stepper, and a charge-coupled device camera .For live-cell imaging, cells were put on ME agarose pads, containing 4% agarose . In expeFor spindle dynamics, stacks of seven planes spaced by 1 µm were acquired for each channel with 200 ms exposure for 561 nm wavelength and 100 ms exposure for 491 nm wavelength, binning one, and an electronic gain of 3 for both wavelengths. For each time-lapse movie, each stack was taken every minute for a duration of 180–240 min.For intensity measurements, stacks of 11 planes spaced by 0.5 µm were acquired for each channel with 100 ms exposure, binning one, and electronic gain of 1 or 3. For each time-lapse movie, each stack was taken every minute for a duration of 90 min.For MT bundle dynamics, stacks of seven planes spaced by 1 µm were acquired for each channel with 100 ms exposure for 561 nm wavelength and 100 ms exposure for 491 nm wavelength, binning one, and an electronic gain of 3 for both wavelengths. For each time-lapse movie, each stack was taken every 5 s for a duration of 30 min.To quantify spores, bright-field images of zygotes were taken at random, and the spores inside the zygotes counted.Analyses were performed by Metamorph 7.8 and ImageJ 1.52i. Maximum projections of each stack were performed for analysis of spindle dynamics and for presentation unless stated otherwise; sum projections were performed for intensity measurements.−1 MBC was added, by measuring the distance between SPBs, from the onset of spindle assembly to spindle breakdown by using a semi-automated MATLAB program.Spindle dynamics were determined by measuring the duration of the mCherry-Atb2 fluorescence signal between Sid4-GFP signals over time or, when 5 µg mlSignal intensity measurements were performed by line scan analysis, reading out the average intensity per pixel, subtracting the background and adding all positive values to obtain the total amount of fluorescence intensity.MT bundle dynamics were approximated by tracing the shape of mCherry-Atb2 signal on a kymograph, and calculating the slopes of the gathered curves to obtain the velocity of MT bundle growth and shrinkage. The origins and the endings of the curves were approximated by extending the curves to the edges of the SPB signal. The movements of the SPB were accounted and corrected for in our measurements. The catastrophe frequency was approximated as a reciprocal value of time the MT bundle spent in elongation.Microscopy images were analyzed with a semi-automated MATLAB program that can find features in microscopy images. After the computer finds the features, they are presented to the user, and the user can manually correct them. In this work, the program was used to detect SPBs and spindles in maximal projections of confocal microscopy stacks. Features are represented as the coordinates of two points (SPBs) or a polygonal chain (spindle). The scripts for the analysis are integrated in a Graphical User Interface, and the complete version of the program is available upon request. We provide the MATLAB scripts for feature extraction, and describe the pipeline of the analysis:Initially, the user segments a cell manually in the relevant frames of the movie. Only the selected frames, and the area inside the segmented region of interest is considered for feature detection for a cell. Features are detected for each of the selected frames.MATLAB'smultithresh. Serial thresholding steps are applied until the area above the threshold contains <10% of the pixels of the region defined by the user as the cell. After that, regions above the threshold are merged using a convex hull algorithm as provided in MATLAB'sbwconvhull and the coordinates of the pixels above the threshold are fit to a first degree polynomial by regression. The resulting line is uniformly sampled such that points are distributed along its length at 1 pixel distance, and constitutes the spindle feature. Once a spindle is found to be >4 μm, spindles are detected using the method explained below.Short spindles (<4 μm) are found by applying a 2 pixel Gaussian blur to the image, and subsequently thresholding applying Otsu's method as provided in 2]. Then,  coordinates are fitted to a first-order polynomial when the cumulative distance along the curve  is <8 μm, and to a third-order polynomial otherwise. The resulting curve is sampled as described previously, and constitutes the spindle feature.This method relies on the fact that long spindles are often aligned with the major axis of the cell. Therefore, the distance, with respect to the major axis of the cell, is very similar in neighboring pixels that belong to the spindle. A maximal projection of the image is made along the major axis of the region defined by the user as the cell; for each projected pixel, the position along the major axis [X], the intensity [I], and distance with respect to the major axis [Y] are stored in 1-dimensional vectors. To keep only the pixels belonging to the spindle, subsequent discarding steps are applied: a moving standard deviation filter of window size 3 pixels is applied to [Y], and positions with a standard deviation value >3 pixels, are discarded. Next, ‘groups’ – denoting a series of consecutive non-discarded pixels – containing <4 pixels are discarded. Pixels for which the intensity in [I] is <20% of the maximum pixel in the cell are discarded. For the remaining groups, if the distance along the major axis [X] between the closest points of two consecutive groups divided by the difference in distance from the major axis [Y] between those two points is >1.5, the group that is furthest from the center of the cell is discarded. The curve defined by [Y] is smoothened by applying a moving median filter of window size 10, resulting in  and , as the sum of Eucledian distances between consecutive points:Spindle length is defined by using the P-values are shown within plots and data sets were defined as significantly different when P<0.05. n values represent the number of cells and are shown inside the plots. All experiments were performed at least three times.All graphs and box plots were generated using GraphPad 8.0 (Prism). All bar graph and line graph data are shown as mean values with error bars representing ±s.e.m. Data in box and whisker graphs were obtained by using the Tukey method; black lines shows the median value. Analysis of statistical significance was performed by using Mann–Whitney or χ² tests. Obtained