This cross-sectional study investigates the use of circulating serum biomarkers to distinguish individuals without diabetic retinopathy from those with sight-threatening diabetic retinopathy. Can circulating serum biomarkers distinguish people with sight-threatening diabetic retinopathy (STDR) from those with no DR?This multicenter cross-sectional study of 538 participants found an incremental benefit of circulating cystatin C beyond the standard clinical variables in discriminating STDR from no DR. Cystatin C outperformed 12 other biomarkers found to be distinguished in STDR in previous research.Results of this study suggest the consideration of circulating cystatin C levels as a triage test in prioritizing people with type 2 diabetes from the community for retinal screening in resource-restricted settings. It is a global challenge to provide regular retinal screening for all people with diabetes to detect sight-threatening diabetic retinopathy (STDR).To determine if circulating biomarkers could be used to prioritize people with type 2 diabetes for retinal screening to detect STDR.This cross-sectional study collected data from October 22, 2018, to December 31, 2021. All laboratory staff were masked to the clinical diagnosis, assigned a study cohort, and provided with the database containing the clinical data. This was a multicenter study conducted in parallel in 3 outpatient ophthalmology clinics in the UK and 2 centers in India. Adults 40 years and older were categorized into 4 groups: (1) no history of diabetes, (2) type 2 diabetes of at least 5 years’ duration with no evidence of DR, (3) nonproliferative DR with diabetic macular edema (DME), or (4) proliferative DR. STDR comprised groups 3 and 4.Thirteen previously verified biomarkers were measured using enzyme-linked immunosorbent assay.1c.Severity of DR and presence of DME were diagnosed using fundus photographs and optical coherence tomography. Weighted logistic regression and receiver operating characteristic curve analysis (ROC) were performed to identify biomarkers that discriminate STDR from no DR beyond the standard clinical parameters of age, disease duration, ethnicity (in the UK) and hemoglobin A1c, inclusion of cystatin C had near-acceptable discrimination power in both countries .A total of 538 participants  were recruited into the study. A total of 264 participants (49.1%) were from India , and 274 participants (50.9%) were from the UK . ROC analysis (no DR vs STDR) showed that in addition to age, disease duration, ethnicity (in the UK) and hemoglobin AResults of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Circulating cystatin-C levels may be considered as a test to identify those who require prioritization for retinal screening for STDR. Therefore, regular retinal screening is recommended, but global coverage of systematic DR screening is a formidable challenge.3 Approximately 80% of people with diabetes live in low- and middle-income countries where resources are restricted.1 Visual impairment owing to STDR is also more common in minoritized ethnic groups.1 Therefore, a paradigm shift in DR screening strategy is required.There are approximately 537 million people living with diabetes globally, the majority of whom have type 2 diabetes.4 These need to be validated in a large cohort in a clinical setting before being proposed as a viable triage tool for retinal screening.4An alternative and more cost-effective risk-based strategy for early identification of STDR may be possible if circulating biomarkers could be used to triage those at risk of STDR for retinal screening. Previous discovery and verification studies have reported a selection of circulating biomarkers with good diagnostic power to identify those with STDR.In this study, we aimed to evaluate previously verified blood biomarkers using enzyme-linked immunosorbent assay (ELISA) for their potential usefulness as indicators of STDR.STROBE) reporting guidelines.This study was approved by the National Research Ethics Service in the UK and the institutional review board of Vision Research Foundation and Aravind Medical Research Foundation in India. All patients provided written informed consent, and the study followed the Declaration of Helsinki. Participants did not receive any compensation or incentives to participate. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology , Black, White, and other . Prior studies have shown that racial and ethnic minoritized groups are disproportionately affected by STDR.1c (HbA1c), estimated glomerular filtration rate (eGFR), and lipids were measured in hospital laboratories. The samples were anonymized before transfer to the laboratories. All laboratory staff were masked to the clinical diagnosis and data and were assigned a study cohort.Routinely collected blood parameters such as glycated hemoglobin AThe selected biomarkers comprised complement factor B (CFB), complement factor H (CFH), serpin A4 , α2 macroglobulin (A2m), cystatin C, thrombin (F2), lipoprotein-associated phospholipase A2 (Lp-PLA2), leucine-rich α2 glycoprotein 1 (LRG-1), 8-hydroxy-2′-deoxyguanosine (8-OHDG), afamin, and apolipoprotein A1 (ApoA1), B (ApoB), and C3 (ApoC3).g to 1300g for 10 minutes. Serum was then aliquoted, frozen, and stored at −80 °C until sample processing in each laboratory. For the UK cohort, all 13 markers were analyzed in a single University College London laboratory whereas for the Indian cohort, 6 markers were analyzed at the Aravind Eye Hospital, Madurai, India, and 7 at the Vision Research Foundation, Chennai, India.Laboratory researchers in both countries followed the same storage and processing instructions. Blood samples were collected in serum separator tubes (BD vacutainer [BD]), allowed to stand for 60 to 120 minutes, then centrifuged at 1100Serum samples were thawed on ice and serially diluted to the required, previously identified, concentration. ELISA procedures, including the establishments of standard curves, were carried out in accordance with manufacturer’s instructions  with diabetic macular edema (DME) or group 4, individuals with type 2 diabetes and PDR with or without DME. DR and DME were diagnosed using fundus photographs and optical coherence tomography, respectively.A sample size of 45 participants with diabetes and no DR was sufficient for modeling STDR outcomes as this reliably distinguished between observed specificity estimates of 60%, 75%, and 90%, having respective 95% CI half-widths of 14.3%, 12.7%, and 8.8%. A sample size of 50 participants allowed for an anticipated 10% of missing data. Assuming that 135 of 150 participants (90.0%) recruited with STDR have complete data, observed sensitivity estimates of 60%, 75%, and 90% for detecting STDR would also be well distinguished, having 95% CI half-widths of 5.1%, 7.3%, and 8.3%, respectively. Sample groups with full data of sizes 135 and 45 participants also provide informative 95% CI half-widths of 0.09, 0.07, and 0.04 for observed areas under the receiver operative characteristic (ROC) curve of 0.6, 0.75, and 0.9, respectively.t test or Mann-Whitney U test for continuous variables and χ2 test or Fisher exact test for categorical variables. Probability weighted comparisons were made between no DR and combined STDR group. Potential multicollinearity was assessed using the variance inflation factor. Weighted logistic regression was used to identify variables that improve detection of STDR relative to no DR, in addition to a (base) model consisting of known risk factors for STDR: age, disease duration race and ethnicity (in the UK), and HbA1c. Owing to the low prevalence of STDR in people with diabetes, recruiting a cohort that is representative of the general population in the proportion with STDR would require a large sample size to ensure there were sufficient events. Therefore, we oversampled STDR groups in our study and used weighted logistic regression to mitigate differences in the sample and population prevalence induced by case-control sampling.6 The diabetes population proportions of the groups were derived as follows: group 2 (no DR), 93.96%; group 3 (NPDR with DME), 5.28%; and group 4 (PDR), 0.76% for the UK from the English Diabetes Eye Screening Programme7 and group 2 (no DR), 94.80%; group 3 (NPDR with DME), 4.45%; and group 4 (PDR), 0.75% for South India from the South India part of the Statistical and Economic Modeling Study of Risk-Based Stratified and Personalized Screening for Diabetes and its Complications in India (SMART) India study,8 after discounting the NPDR without DME group, not recruited in this study. Probability weights wi for each disease group, were calculated as iτ/iy, the ratio of the population proportion (iτ) and the sample proportion (iȳ). Variables that reached statistical significance in the adjusted analysis were introduced into a forward stepwise selection routine with an entry criterion of α = 0.1, with age, disease duration, race and ethnicity (in the UK), and HbA1c forced into the models. Loge transformation was used for variables serum creatinine, C-reactive protein, triglycerides, A2m, 8-OHDG, ApoA1, ApoB, ApoC3, ApoB/ApoA1, LRG-1, and cystatin C for logistic regression analysis, to avoid estimating misleading associations from influential outlying observations. Odds ratios (ORs), therefore, are interpreted in relation to relative percentage increase in the biomarker rather than per absolute increase in the measured units for other biomarkers.Data were summarized by outcome group and by country, using mean (SD) or median (IQR) for continuous variables and number (%) for categorical variables. Statistical differences between no DR (group 2) and no diabetes (group 1) as well as between no DR (group 2) and individual STDR groups 3 and 4 were compared using the 2) as elevated cystatin C levels may be attributable to diabetic kidney disease rather than STDR. All 2-tailed P values < .10 were considered statistically significant. Statistical analysis was carried out in RStudio, version 3.6.3 (RStudio)9 and Stata MP, version 15 (StataCorp).10The final models were summarized using ORs with 95% CIs and the ROC curve. Sensitivity analysis was carried out to compare the area under the curve (AUC) of closely related biomarkers serum creatinine and cystatin C. In addition, cystatin C levels were compared between STDR (groups 3 and 4) and no DR (group 2) in patients with normal kidney function  were collected from October 22, 2018, to December 31, 2021, exceeding the minimum requirement for 500; however, in the UK, 91 biomarker samples were lost owing to freezer failure  and LRG-1 . The base model consisting of variables age, duration, race and ethnicity, and HbA1c contributed an AUC of 0.735 . The addition of cystatin C to the base model yielded an AUC of 0.779  and LRG-1 to the base model yielded an AUC of 0.763 .Adjusted logistic regression results for demographic, clinical, and biomarker data are shown in P = .003), afamin , cystatin C , and Lp-PLA2  . The addition of cystatin C to the base model contributed an AUC of 0.696  and the base model plus CFB contributed an AUC of 0.665 .Biomarkers that were found to be associated with STDR (groups 3 and 4) relative to no DR (group 2) in the adjusted analysis were as follows: CFB  . Interes1c, cystatin C, and LRG-1), the variance inflation factor ranged from 1.1 to 1.2 in the UK. In India, the variance inflation factor ranged 1.1 to 1.6 for variables age, duration, HbA1c, cystatin C, afamin, A2m, Lp-PLA2, and CFB, which were variables considered for inclusion in the multimarker panel, indicating that multicollinearity is unlikely to cause problems for our analysis.In those variables that reached significance in adjusted analysis  on top of variables in the base model. In India, additional biomarkers included cystatin C and CFB, with a combined AUC of 0.735  , we would need to screen 704 patients out of 1000 to detect 42 out of 52 STDR cases, with approximately 97% of low-risk patients correctly identified as having no STDR.2 or greater (63 in the UK [33 STDR events] and 61 in India [38 STDR events]). In patients with an eGFR of 90 mL/min/1.73m2 or greater, the presence of STDR correlated with elevated cystatin C levels in the UK  and similarly in India .As both cystatin C and serum creatinine had comparable AUC, cystatin C (log) was substituted with serum creatinine (log). In the UK, only 205 patients with 137 STDR events had available data to use serum creatinine instead of cystatin C in the models. The base model with serum creatinine instead of cystatin C, yielded an AUC of 0.779, and when combining with LRG-1, the AUC was 0.791  and STDR (groups 3 and 4). Comparing the STDR and no DR groups in India, CFB, A2m, afamin, cystatin C, and Lp-PLA2 were significantly different at the α = 1% threshold. Owing to interlaboratory variations in some of the biomarkers investigated, we opted to include only the most stable biomarker in the strategy with near-acceptable discrimination in both study cohorts.1c in the UK, the analysis showed acceptable performance, with an AUC of 0.779 compared with an AUC of 0.735 without cystatin C. In India, the model with age, duration, and HbA1c had performance closer to chance, with an AUC of 0.552, and adding cystatin C had close to acceptable performance with an AUC of 0.696. The validity of this strategy was similar in the UK and India; therefore, results suggest that this biomarker was useful in both countries.15When cystatin C levels were added to age, duration of diabetes, race and ethnicity, and HbA20Both models achieved adequate sensitivity while preserving test specificity to an extent; however, the UK models had higher specificity across all tested thresholds. This might be partly attributable to the duration of diabetes not being as accurate in detecting STDR in India unlike the UK, where this was one of the strongest indicators of STDR. Nonetheless, our results show stability across both data sets. Several studies done globally have demonstrated an association between cystatin C and DR.21 Third, in contrast to many other published studies, which often use mass spectrometry of plasma,12 we opted for ELISA analyses of serum to remain close to what is routinely available in a health care setting.22 Fourth, data for cystatin C, used in our final model, did reflect that seen in the literature.19This study had several strengths. First, both the UK and India models achieved close to acceptable discrimination. Second, the models relied mostly on routinely collected data, except for cystatin C. However, a point-of-care cystatin C biosensor is feasible to produce, will not require skilled workers to perform, and may be accessible as a web-based application for self-monitoring by patients.1c kits are already available for community screening.23 Fourth, validation of a cystatin C point-of-care kit is needed before use in a health care– or community-screening setting. Fifth, patients with NPDR but without DME were not recruited into this study, and therefore, statistical models may not generalize to all patients with diabetes or represent population-level risk of STDR. Sixth, serum cystatin C has also been shown to be an early marker of diabetic kidney disease,24 but our results showed that cystatin C remained elevated in STDR in a subgroup of patients with normal eGFR.However, there were several limitations. First, we were unable to pool the samples from UK and India owing to differences in the distribution of some biomarkers. More studies are required to understand intercountry variations in the profiles of these biomarkers. Second, further validation in a cohort representative of the real-world prevalence of STDR and not oversampled for cases is needed. Third, as these markers were analyzed in stored and freeze-thawed serum only, further validation of our prescreening strategy should be undertaken in a community screening setting using finger-prick blood test. Notably, similar low-cost point-of-care HbAResults of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Although retinal imaging is the criterion standard for DR screening and should be advocated for all patients with diabetes on an annual or biennial basis, this is not always feasible in low- and middle-income countries. In such low-resource settings, prescreening models with cystatin-C may potentially be used to identify those who need prioritization for retinal screening.


We observe anomalous terahertz emission in photo-excited high- 2-xBaxCuO4 family, for which this effect is expected to be forbidden by symmetry. We find that emission vanishes for compounds in which the stripes are quasi-static but is activated when c-axis inversion symmetry is broken by incommensurate or fluctuating charge stripes, such as in La1.905Ba0.095CuO4 and in La1.845Ba0.155CuO4. In this case, terahertz radiation is emitted by surface Josephson plasmons, which are generally dark modes, but couple to free space electromagnetic radiation because of the stripe modulation.The interplay between charge order and superconductivity remains one of the central themes of research in quantum materials. In the case of cuprates, the coupling between striped charge fluctuations and local electromagnetic fields is especially important, as it affects transport properties, coherence, and dimensionality of superconducting correlations. Here, we study the emission of coherent terahertz radiation in single-layer cuprates of the La For exa3, is based on optical rectification, where the second-order nonlinear optical susceptibility causes a time-dependent electrical polarization , with a critical temperature of 38 K (phase diagram in 2-xBaxCuO4 (LBCO), for which superconductivity coexists with charge stripes . These were focused at normal incidence onto an ac-oriented sample surface on an ∼ 500 μm spot. The emitted THz pulses were collimated with a parabolic mirror and refocused on a 1-mm-thick ZnTe crystal to perform electro-optic sampling directly yielding THz electric field traces in time domain.The main result of our experiment is summarized in In optimally doped LSCO E, the THspectrum I. A simispectrum F and J, On the other hand, in LBCO 15.5% ,In the compound with incommensurate, relatively strong stripes, i.e., LBCO 9.5%, the THz emission amplitude was even higher than LBCO 15.5% and greater by a factor of observed H, corresobserved L. The frSI Appendix).The rest of the analysis in this paper is focused on LBCO 9.5%, which yielded the largest signal and highest coherence. We verified that the emission was entirely polarized along the out-of-plane crystallographic axis and could be induced only for a pump polarization aligned along the same direction  , 38. TheIn interpreting our results, we first note that in a centrosymmetric cuprate, impulsive excitation of Josephson plasmons is forbidden by symmetry. Josephson plasma modes are in fact symmetry-odd (infrared-active), while impulsive photoexcitation couples only to totally symmetric modes . As discx ≳ 1/8 , 34, whix ≳ 1/8 A 44). A. Ax ≳ 1/x ≳ 1/8 , resultix ≳ 1/8 B.shift current (bulk Josephson plasma polariton, sustained by tunneling supercurrents oriented in the surface Josephson plasmon, also sustained by plasma oscillations in the Once inversion symmetry is broken, electromagnetic emission at a frequency  current , 44 at tRadiation from bulk plasma polaritons C, exciteμm , would brequency B.Coherent narrowband emission by surface Josephson plasmons is instead more likely. Although the dispersion of these modes lies below the light cone and, hence, they are not expected to radiate into vacuum D, we arg47Umklapp shift current, In the right panel of In summary, we have reported the observation of coherent THz emission just below the Josephson plasma frequency in cuprates for which the superconducting state coexists with stripes. We assigned this effect to the excitation of surface Josephson plasmons, which become Raman active due to the breaking of inversion symmetry induced by the stripes and can radiate out thanks to the backfolding of their dispersion curve onto the light cone. Based on these findings, the characterization of coherent THz emission emerges as a sensitive method to unveil broken symmetry states, which may not be detectable with other conventional techniques. Moreover, the absence of THz emission in LBCO 11.5%, where the stripes are more robust and quasi-static, may suggest a qualitative difference in the nature of charge and spin order between compounds that are in the vicinity of the commensurate 1/8 doping and those that are far from it.Supplementary File


BackgroundSince posterior cervical fixation with lateral mass screws was introduced in 1979, multiple techniques have been described in the literature. However, no study to date has determined whether pre-drilling all lateral masses prior to screw insertion has a benefit over the traditional sequential drilling and screw insertion on the alignment of the screw-rod construct. This study sought to determine the efficacy and efficiency in achieving alignment with a novel pre-drilling technique compared to the traditional sequential drilling technique. The authors hypothesized that the novel pre-drilling technique could be applied more quickly and precisely than the traditional sequential drilling technique.MethodsEight cervical spine sawbones models were utilized to place 64 lateral mass screws by two surgeons. The pre-drilling technique was utilized to place 32 screws in four models, and the sequential drilling technique was utilized to place the 32 screws in the remaining four models. In the traditional sequential drilling technique, each lateral mass underwent screw tract preparation and insertion before proceeding to the subsequent vertebra. In the pre-drilling technique, all lateral masses were marked and drilled sequentially before screw placement. CT imaging with 3D reconstructions was generated for all models. Variability in screw placement and time taken to fully instrument the models were compared. ResultsThe mean time to completion of the pre-drilling technique was 337 ± 22 seconds compared to 490 ± 22 seconds with the traditional technique (p<0.01). There was a significantly higher variability in the coronal plane within the traditional group between C5 and C6 compared to other adjacent vertebrae (p<0.05). There was no significant difference in the start point variability and the overall tightness of line fit between the techniques.ConclusionsOur study suggests that a novel pre-drilling technique for lateral mass screw insertion may be more efficient and reliable than the traditional sequential drilling technique. In addition, this technique may reduce the need for rod contouring or additional implants to optimize the alignment of cervical instrumentation. However, further clinical studies are necessary to validate the potential clinical and radiologic benefits of this described technique. Posterior cervical instrumentation constructs can safely realign the spinal column while achieving arthrodesis in various spinal disorders, including trauma, tumor, spondylosis, infection, inflammatory/rheumatologic disease, and other conditions . PosteriMaximizing surgical efficiency while minimizing screw position variability are essential concepts for achieving optimal outcomes after posterior cervical instrumentation. Recent studies have found significant associations between longer operative time and postoperative length of stay, non-home discharge, and transfusion requirements after cervical spine surgery ,8. FurthAlthough multiple screw placement techniques have been described in the literature, to our knowledge, no study to date has determined whether pre-drilling all lateral masses prior to screw insertion is beneficial over sequential drilling and screw insertion in regards to surgical efficiency and on the screw-rod construct alignment ,12-14. TThe technique chosen for posterior cervical lateral mass fixation and screw trajectory was patterned after the technique that is used in the senior author’s practice . The aforementioned technique itself is an adaptation of the technique, which has been described and adopted by many spine surgeons ,14. An oEight prone-positioned cervical spine sawbones models  were mounted onto a foam stabilizer and were categorized into four groups. The two techniques were each performed in two groups; one group was timed, and the other untimed. Technique 1 represents the traditional, sequential drilling and screw placement technique; Technique 2 represents the senior author’s preferred technique of pre-drilling all lateral mass screws pilot holes prior to screw insertion. TechniqueNIH.com) program. The instrumented models were then imaged with CT, and 3D reconstructions were generated. Measurements for the screw start points were calculated as distance (in mm) from the pilot holes to the spinous process (representing the midline) and in relation to each other. The variability of screw start point placement in the transverse plane was compared between techniques by measuring the difference in the start point - spinous process distance across levels. The number of seconds taken to fully instrument C3-C6 bilaterally was also measured between techniques.First, the midsagittal and mid-axial intersection of the lateral mass was identified. Then, a marking was made using a marking pen at 1 mm medial and 1 mm caudal to the center of the quadrangular dorsal surface of the lateral mass. Next, a 2 mm pilot hole was created at this mark with a trajectory directed to the center of the lateral mass using a high-speed 3 mm x 3.8 mm burr. The 2.4 mm drill bit was then placed within the burr hole directed at the superolateral corner of the lateral mass. At this point, the techniques diverge: in Technique 1 , the lateral mass of the first vertebra underwent pilot hole burring, followed by drilling, tapping of the screw tract, and then screw insertion, before moving to the subsequent vertebra. In Technique 2 (pre-drilling), all lateral masses were first marked before the pilot hole was created. The burr holes were then created sequentially from cranial to caudal. Then the screw tracts were drilled sequentially in the same fashion. Finally, the tract was tapped, and a screw was placed in each lateral mass, moving to the subsequent/caudad vertebra. Depuy-Synthes Synapse 3.5 mm x 14 mm polyaxial screws  were inserted into the lateral masses of the C3, C4, C5, and C6 vertebrae of all models.All lateral mass screws were placed, and digital photographs of the models with a reference marker were taken in the coronal plane. The specimens were randomized by another blinded investigator prior to photographing and measuring. A straight line between the center of the screw holes at the C3 and C6 lines was made, and a similar line was extrapolated from a straight line between the C4 and C5 levels using the Image J software  x 2 (technique) analysis of variance followed by a Tukey's post hoc test was performed. Additionally, a sum of squares test was performed to determine and compare the overall tightness of line fit from C3-C6 for both techniques. Finally, a student's T-test was used to compare procedure completion time between techniques. Type I error was set at α = 0.05 for all analyses. For all detected pairwise differences at p<0.05 and pairwise comparisons approaching significance at p<0.1, effect size (ES) is presented as Cohen's D statistic [Cohen's d = (Mean2 - Mean1) / SDpooled] whereby values are interpreted as follows: ES < 0.1, negligible; ES 0.1-0.3, small; ES 0.3-0.5, moderate; ES 0.5-0.7, larger; and ES > 0.7, very large.Type III tests of fixed effects revealed an interaction of method and vertebral segment (p<0.05). Following post-hoc analysis, no significant difference was observed between techniques (p>0.05). However, the traditional sequential drilling technique demonstrated greater variability in the coronal plane between C3 and C4  and C5 and C6  . Analysis of the sum of squares revealed no difference in the tightness of line fit between the techniques. The mean time to completion of the pre-drilling technique was 337 ± 22 seconds compared to 490 ± 22 seconds with the traditional technique (p<0.01)  . The scrRecent research suggests that surgeons have difficulty precisely matching these expected angles with the naked eye ,2. WhileWe compared the absolute time needed to place bilateral lateral mass screws with the proposed technique versus a traditional method. We found that the proposed technique could be performed quicker due to minimizing unnecessary technical moves. To our knowledge, no previous study has compared lateral mass screw techniques in terms of operative time. It is well-known through the spine and general surgical literature that increased operative time is associated with increased complications such as infection, venous thromboembolism, pulmonary compromise, and hospital readmission ,8,18,19.In the current study, we also sought to demonstrate that the proposed technique resulted in the more consistent placement of screws along a straight paramedian line. We believe that achieving this goal reduces the need for repeated contouring of rods or using additional implants, particularly in constructs that span the occipital-cervical or cervicothoracic junctions. It has been demonstrated in biomechanical and clinical studies that over-manipulation of rods can contribute to fatigue failure -11. FurtThere are limitations in the current study that we acknowledge. While sawbones models are anatomically accurate simulations that have been used to augment surgical skills, they do not account for variations in soft tissue or structural anatomy nor dynamic factors that one encounters when performing cadaver or live patient operations ,21. In aOur study suggests that a novel pre-drilling technique for lateral mass screw insertion may be more efficient and reliable than the traditional sequential drilling technique. In addition, this technique may reduce the need for rod contouring or additional implants to optimize the alignment of cervical instrumentation. However, further clinical studies are necessary to validate the potential clinical and radiologic benefits of this described technique.


In ecology and evolution, such terroir  is expected to play an important role in shaping phenotypic traits. Just how important is the pure spatial effect of terroir  in comparison to temporal variation , and the interaction between space and time ? We answer this question by analyzing beak and body traits of 4388 medium ground finches (Geospiza fortis) collected across 10 years at three locations in Galápagos. Analyses of variance indicated that phenotypic variation was mostly explained by site for beak size (η2 = 0.42) and body size (η2 = 0.43), with a smaller contribution for beak shape (η2 = 0.05) and body shape (η2 = 0.12), but still higher compared to year and site‐by‐year effects. As such, the effect of terroir seems to be very strong in Darwin's finches, notwithstanding the oft‐emphasized interannual variation. However, these results changed dramatically when we excluded data from Daphne Major, indicating that the strong effect of terroir was mostly driven by that particular population. These phenotypic results were largely paralleled in analyses of environmental variables  expected to shape terroir in this system. These findings affirm the evolutionary importance of terroir, while also revealing its dependence on other factors, such as geographical isolation.The term  The term terroir is used in viticulture to emphasize how the biotic and abiotic characteristics of a local site influence grape physiology and the properties of wine. In ecology and evolution, such terroir  is expected to play an important role in shaping phenotypic traits, but how important is the pure spatial effect of terroir in comparison to temporal variation, and the interaction between space and time? We answer this question by analyzing beak and body traits of 4388 medium ground finches (Geospiza fortis) collected across 10 years at three locations in the Galápagos Islands. Finally, (3) spatial and temporal differences in traits between population A and B can increase/decrease depending on their location and how long have they been monitored. Here, we explore these questions by analyzing a 10‐year dataset of environmental features and phenotypic traits in three populations of Darwin's finches. We then compare our results to those from other classic systems in evolutionary biology. We close with a discussion of how the concept of terroir might be useful in helping to reframe and reinvigorate considerations of how temporal and spatial effects contribute to ecology, evolutionary biology, and eco‐evolutionary dynamics.1.1Terroir is likely to be very important for Darwin's finches in the Galapágos because different islands, and even different sites within an island, can show dramatic differences in species composition and—for some species—striking variations in morphological traits  across a 10‐year period. We first use Analysis of Variance (ANOVA) to partition the variation in environmental variables  into the main effect of site, the main effect of year, and the interaction between site and year. We then use univariate and multivariate ANOVAs for a similar partitioning of beak and body trait data. Finally, we use phenotypic trait trajectory analyses (PTA) to explore the contributions of space (site) to temporal changes in multivariate trait means. We conduct these analyses first using all three populations: the small island of Daphne Major and two sites (Academy Bay and El Garrapatero) on the large island of Santa Cruz. Then, because Daphne Major appears to be a special case, we repeat the analyses using only the two sites on Santa Cruz.These effects and their relative impacts have not been formally quantified and compared for Darwin's finches because no study to date has quantified and compared both spatial variation (multiple sites) and temporal variation (multiple years) in the same analysis. We do so here by compiling annual environmental and trait data for three populations of the medium‐ground finch  and from two lowland sites on the island of Santa Cruz: Academy Bay  and El Garrapatero  Figure . Academy2.2We obtained rainfall and spectroradiometric indices of vegetation for the 10 years of our study, 2003–2012 Figure . Daily rRemote sensing data were used to obtain four indices associated with vegetation cover over the 2003–2012 period. More specifically, from the Moderate Resolution Imaging Spectroradiometer (MODIS) database  and 1 km2 (for LAI and FPAR) area directly north of the Baltra Airport, which is nearby to DM and has similar physical characteristics as explained above.For AB and EG, NDVI and EVI were obtained for an area of 250 m × 250 m , and LAI and FPAR were obtained for an area of 1 km2.3G. fortis) each year from 2003 to 2012 in the three study sites . In all cases, the birds were captured with mist nets and then banded with uniquely numbered metal leg bands to ensure that individuals were not sampled multiple times. Each bird was inspected and classified—based on plumage, beak color, and the presence of a brood patch—as a juvenile, male, or female  as suggested in Cohen  were used to examine how spatial variation (main effect of site), temporal variation (main effect of year), and the interaction between these two factors explained variation in rainfall and vegetation indices. Type III Sums of Squares were used given the presence of the site‐by‐year interaction term in our models. The comparisons that could be made were (1) Baltra (for DM) versus AB for log‐transformed daily rainfall, and (2) Baltra (for DM) versus EG versus AB for the monthly average of vegetation indices . Additionally, the same analyses were performed after excluding Baltra (DM) so that we could test the extent of variation between two sites (AB and EG) on the same island. Effect sizes for each of these factors were quantified using partial eta square  separately for beak traits  and then for body traits . PCA based on the covariance matrix was performed for beak traits, following previous analyses  of spatial variation (site), temporal variation (year), and their interaction. Sex  was included in the models as a fixed effect. Juveniles were excluded given that their beak and body traits are still developing , beak shape (PC2 of beak traits), body size (PC1of body traits), and body shape (PC2 of body traits) were analyzed using separate linear fixed‐effect models with Type III ANOVAs applied to quantify the relative contributions , and then excluding DM.Because the above analyses relied on PCA‐restructured trait (co)variances, as has been typical for research on finches, we also analyzed the original trait measurements in multivariate analyses of variance (MANOVA) using the “Pillai” test, which accounts for our varying sample sizes. These analyses were run separately for beak and body traits, and effect sizes were again quantified as partial 2.4.3L) and directions (angles θ) in a pair‐wise fashion . Trajectory length comparisons inform the difference among sites in the amount of among‐year multivariate trait variation along primary axis of interannual change. Trajectory direction comparisons inform the difference among sites in the multivariate orientation of those primary axes of interannual change. See Adams and Collyer  among sites and years from the reported F‐values and the degrees of freedom associated to them following Cohen , and all of these indices were correlated with total annual rainfall at each site . Not surprisingly, then, all indices yielded similar insights into terroir.Yearly averages of the four spectroradiometric indices were strongly correlated with each other at each site  by approximately 70% for the main effect of site, increased  by approximately 17% for the main effect of year, and decreased by approximately 20% for the site‐by‐year effect. These overall reductions in the relative importance of site suggests that spatial consistency across years across our entire sample is mainly driven by substantial differences between DM and the two Santa Cruz sites (AB and EG). However, it is important to note that site effects were still strong when comparing some vegetation indices within Santa Cruz Island, between EG and AB Table .3.2G. fortis at DM as compared to AB and EG . PCA‐based analyses showed strikingly smaller beaks, pointier beaks, smaller bodies, and larger wings in G Figure . FurtherDM Table . During DM Table , with exDM Table . Beak shDM Table .η2 = 0.42) and body size (η2 = 0.43), both of which were much larger than the corresponding effect for beak shape (η2 = 0.05) and body shape (η2 = 0.12). The main effect of year and the site‐by‐year interaction were of similar magnitude in all cases  and interactions of regional variation with site‐specific factors (site‐by‐year interaction). These results follow those seen for rainfall and vegetation indices in that the main contribution of terroir lies in generating site‐specific phenotypic differences that mainly persist across year.Echoing the above‐noted differences between sites, ANOVA and MANOVA analyses indicated that the main effect of site explained most of the variation, followed by the main effect of year and then the site‐by‐year interaction Table . The larη2 = 0.4, both ofG. fortis dataset revolves mostly around the beak size and body size (but not shape) of DM birds relative the Santa Cruz (AB and EG) populations.After removing DM from ANOVA and MANOVA analyses of finch morphology, overall effect sizes for site decreased (relative to the models with DM) by 95%, year effects increased by 28%, and site‐by‐year effects decreased by 50% Table . Thus, vmagnitude (ΔL) and direction (θ) of temporal variation in beak and body traits further illustrated the importance of terroir (effect of site) in G. fortis traits. Specifically, for beak traits, average differences were greater in the direction of trajectories compared to their magnitude, which indicates the effect of terroir in creating divergent phenotypic trajectories  revealed differences among sites in the length and direction of the multivariate trajectories for mean beak and body traits Table . That ises Table . For bodes Table . When paes Table . These r3.3G. fortis in comparison to other systems, but only for the presence of the DM population.The importance of terroir differed among traits and study systems Figure . Overall4terroir is intended to highlight the importance of local biotic and abiotic conditions in shaping organismal attributes. One way that terroir could play out for Darwin's finches would be differences among sites in finch traits and finch community composition. Indeed, spatial differences among finches were the focus of early studies on this group . Most prominently, our analysis revealed a very strong signature of “terroir”—that is, temporal changes in beak and body traits were typically small relative to the magnitude of phenotypic differences among sites. Moreover, these patterns of trait variation closely mirrored the strong and temporally consistent differences among sites in climate  and vegetation indices   Table . These v4.1Terroir could manifest as temporally consistent differences among sites  or as site‐specific temporal changes . Our results mainly fall into the first category; that is, consistent differences among sites tend to be more important than site‐specific temporal changes. This outcome likely reflects physical features of the sites that generate consistent differences in rainfall, which generates consistent differences in plants, which generate consistent differences in finch traits.2 with a peak elevation of 120 m, and it falls in the rain shadow (given the prevailing winds) of Santa Cruz  often eaten by large morphs of G. fortis on Santa Cruz  and El Garrapatero (EG)—are both located in the lowlands and are more similar to each other—in all respects—than either site is to DM. For instance, average values for size than for beak and body shape, the later mainly being relative wing length  and EG (~25 km away)  might reflect their more extreme and challenging environment. This hypothesis could be tested by analyzing phenotypic variation among additional populations in relation to average climate and vegetation measures. a Grant,  could beG. fortis on the two islands is not that Santa Cruz lacks small G. fortis, but rather that DM lacks large G. fortis: that is, the range of beak sizes is greater on Santa Cruz, especially at the large end of the distribution , which could favor smaller G. fortis individuals who could take advantage of the smaller seeds that G. fuliginosa would otherwise eat. Further, the colonization and rapid increase of the large ground finch population (Geospiza magnirostris) on DM precipitated a character‐displacement shift toward even smaller beak sizes  than those on Santa Cruz.Third, composition of the finch community on DM differs from that at AB and EG, which could precipitate divergent patterns of selection. For starters, only DM lacks the small ground finch  were in similar lowland arid habitats, whereas Finally, we note that the small interannual effects (relative to site effects) in our study system could be due to the fact that Darwin's finches are long lived, and that beak size is very strongly genetically determined ; formal analysis (lead); investigation ; methodology ; software ; validation ; visualization ; writing – original draft (lead); writing – review and editing (lead). Joost Raeymaekers: Conceptualization (lead); data curation ; formal analysis ; investigation ; methodology ; project administration (lead); validation ; writing – original draft ; writing – review and editing . Luis F. De León: Investigation ; methodology (supporting); writing – review and editing . Jaime Chaves: Investigation ; methodology ; writing – review and editing . Diana Sharpe: Investigation ; methodology ; writing – review and editing . Sarah Huber: Investigation ; methodology ; writing – review and editing . Anthony Herrel: Investigation ; methodology ; writing – review and editing . Bieke Vanhooydonck: Investigation ; methodology ; writing – review and editing . Kiyoko Gotanda: Formal analysis (supporting); investigation ; methodology ; visualization (supporting); writing – review and editing . Jennifer Koop: Investigation ; methodology ; writing – review and editing . Sarah A. Knutie: Investigation ; methodology ; writing – review and editing . Dale Clayton: Investigation ; methodology ; writing – review and editing . Jeff Podos: Investigation ; methodology ; writing – review and editing . Andrew Hendry: Conceptualization (lead); funding acquisition (lead); investigation ; methodology ; project administration (lead); supervision (lead); validation (lead); writing – original draft ; writing – review and editing (lead).The authors declare no conflicts of interest.Tables S1‐S2Click here for additional data file.


Sweat aggravates atopic dermatitis (AD). In patients with AD, type-I hypersensitivity to sweat may be shown by histamine release from patients’ basophils in response to the semi-purified sweat antigen (QR), and the presence of specific immunoglobulin E (IgE) binding to MGL_1304, the component of QR. However, there has been no information on the immunological changes of type-I hypersensitivity to the sweat antigen in patients with well-controlled AD using topical corticosteroids (TCSs) and/or biologics as treatments.Histamine-releasing tests using patients’ basophils and QR and the detection of serum IgE against MGL_1304 and mite allergen Der f 1 were performed in patients with AD who were well controlled by topical TCS with/without dupilumab for 53–96 weeks.In total, 14 patients were enrolled. Seven patients received TCS therapy alone (TCS group), and seven patients received TCS with dupilumab therapy (dupilumab group). In all participants, the level of specific IgE against MGL_1304 decreased after treatments, but histamine release from basophils in response to QR did not show a statistically significant reduction; rather, it increased. In the dupilumab group, all changes in histamine release induced by QR (increase), the IgE level against MGL_1304 (decrease), and that against Der f 1 (decrease) were statistically significant, whereas the TCS group showed no significant change in any of them.The well-controlled condition for 53–96 weeks resulted in no reduction of the hyperreactivity of basophils against in patients with AD, even with the treatment with dupilumab. This study suggests persistent basophil hyperreactivity to sweat antigen over a year or longer. Atopic dermatitis (AD) is a common chronic inflammatory skin disease, which is characterized by pruritic, eczematous lesions with the fluctuation of remission and relapse . It is oMalassezia globosa, has been identified as the histamine-releasing component in QR  or 10 ng/ml QR at 37°C for 40 min. After incubation, the cells and supernatant were separated by centrifugation and mixed with HClO4 to a final concentration of 2.5% to denature excess proteins. After centrifugation, the amounts of histamine extracted in the supernatant were measured using high-performance liquid chromatography (HPLC). The HPLC system consisted of an LC-20A  using a VP-ODS column  with an RF-10Axl fluorescence detector  operating at an emission wavelength of 440 nm and an excitation wavelength of 360 nm. The percentage of histamine release was calculated using the ratio of the amount of histamine in the supernatant to the total amount of histamine in the supernatant and cells. For analysis, the value of histamine release against the sweat antigen was normalized by the maximum release of histamine induced by anti-IgE, namely, (net % of histamine release induced by QR)/(net % of histamine release induced by anti-human IgE)×100 (%).QR was prepared by concanavalin A chromatography, anion-exchange chromatography, and reverse-phase chromatography in reference to histamine release activity from the basophils of patients with AD . HRT witM. globosa  in PBS at 4°C overnight. After blocking, the wells were incubated with 100 µl of patients’ sera diluted 1:10 with 10% blocking one solution at room temperature for 2 h. For a standard reaction in each experiment, the wells were also incubated with the serial dilution of the standard serum with high titer to Der f 1. The standard serum was obtained from a patient with AD sensitized to Der f 1. The subsequent method was the same as the ELISA for MGL_1304. The concentration of Der f 1 IgE was calculated in reference to the standard curve.The changes of various parameters between E1 and E2 were calculated by the subtraction of values at E1 from those at E2. The statistical analysis was performed by GraphPad Prism . A p-value of less than 0.05 was considered significant. Groups were compared by using the Wilcoxon matched-pairs signed rank test for paired samples. Correlation was tested with the Spearman’s rank test.Candida albicans, which belongs to another fungal genus  were recruited. Their clinical characteristics are shown in al genus . HoweverThe leukocyte fraction in blood was used to analyze histamine release from basophils in response to the sweat antigen. This fraction contained 0.48%–2.11% basophils, while the magnetic isolation of the basophil population achieved a basophil purity of 83.9%–93.6% by negative selection (n=4)   at least for histamine release to QR    but not in the dupilumab group  .To explore reasons why the hyperreactivity of basophils against QR showed no reduction in spite of the decrease of IgE against MGL_1304, we analyzed the relationship of histamine release in response to QR to the length of periods under a well-controlled disease (IGA ≤2), net histamine release in response to the anti-human IgE antibody, and total serum IgE level. There was no significant correlation of the change of histamine release in response to QR to the length of periods under a well-controlled disease  was not attenuated in the period of a well-controlled disease with the mean interval of 69 weeks (53–96 weeks) regardless of the use of dupilumab. Patients in the dupilumab group showed a significant reduction of specific IgE against MGL_1304 and Der f 1, whereas those in the TCS group did not. Thus, the suppression of the Th2 pathway by dupilumab reduced both the level of IgE against the sweat antigen and that against the mite antigen but did not result in a decrease of reactivity of basophils in response to the antigen.via gastrointestinal uptake.The reduction of basophil hyperreactivity has not been observed in sweat allergy in this study but described in a type of food allergy. A previous study on the basophil hyperreactivity to hydrolyzed wheat (Glupearl 19S) sensitized by facial soap revealed a negative conversion of the basophil reactivity over the time of a year or longer by avoiding exposure to the antigen , suggestvia the high-affinity IgE receptor, resulting in the increase of histamine release in response to QR. The recovery of impaired basophil function during the time between two evaluations may explain a discrepancy between the change of antigen-specific IgE levels and that of histamine release in response to the sweat antigen. The recovery of basophil function may be partially associated with decrease of total serum IgE because most patients with decreased total IgE showed increased histamine release against anti-human IgE .TN, ST and MH designed the study. TN, ST, MH, KI, YY, and ToK wrote the manuscript. KI, AT, HM, SM, YY, TaK and ToK contributed to data collection. TN and ST performed the statistical analysis and interpretation of the results. All authors read and approved the final manuscript.This study was supported by AMED under Grant Number JP19ek0410059 (ST and MH) and by JSPS KAKENHI under Grant Number JP21K08302 (ST), JP21K08346 (MH), and JP20K17319 (KI).TN has received a research grant from Sun pharma and Kyowa-Kirin. ST has received a research grant from Sanofi, Maruho, and Taiho Pharma and an honorarium from Novartis. MH has received a research grant from GlaxoSmithKline, Kaken Pharmaceutical, Mitsubishi-Tanabe, Novartis, Sanofi, and Taiho Pharma, honorarium from Ely-Lilly, Kaken-Pharmaceutical, Kyowa-Kirin, Mitsubishi-Tanabe, MSD, Novartis, Sanofi, Taiho Pharma, Teikoku seiyaku, and Uriach. AT has received a research grant from Maruho, Sanofi, Eisai, and Mitsubishi Tanabe Pharma and an honorarium from Maruho, Torii Pharma, Mitsubishi Tanabe Pharma, Sanofi, and Taiho Pharma.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments. In this study the authors report that Caldesmon controls force-balance and architecture of stress fibers through dynamic cross-linking of actin and myosin filaments. Caldesmon depletion led to consequent problems in cell morphogenesis, motility and mechanosensing. Myofibrils are comprised of regular, repetitive patterns of the force-generating and load-bearing devices, called sarcomeres. The force for muscle contraction is generated by ATP-dependent sliding of bipolar myosin II filaments along a bipolar array of actin filaments within the sarcomeres. Muscle contraction is activated by Ca2+ released from the sarcoplasmic reticulum through the troponin–tropomyosin (Tpm) complex. Ca2+-binding to troponin induces a conformational change, which shifts the position of Tpm along the major groove of the actin filament to allow actin–myosin interactions and muscle contraction3. Two structures within the sarcomeres, the Z-disc and the M-band, anchor actin and myosin II filaments to an elastic filament system that is composed of giant protein titin. The Z-disc harbors α-actinin cross-linked antiparallel actin filaments from the adjacent sarcomeres and several associated proteins5. M-band at the center of the sarcomere is composed of a hexagonal array of proteins including myomesins that position the myosin II filaments to the middle of the sarcomere, and are hence critical in controlling the force imbalances during muscle contraction6.Contractile actomyosin bundles, composed of thin actin filaments and thick myosin II filaments, are key force producers and mechanosensors in diverse animal cell types and tissues. They include myofibrils of striated muscles, contractile filaments of smooth muscles, and stress fibers of non-muscle cells11. However, smooth muscle and non-muscle cells lack many key components of striated muscle myofibrils, such as proteins of the M-band. Therefore, how the organization of actomyosin bundles within contractile units is maintained, and how the force imbalances during contraction are controlled in smooth muscles and non-muscle cells have remained elusive2. Moreover, stress fibers and smooth muscle actomyosin bundles do not express the troponin complex, and thus the mechanisms controlling their contractility are incompletely understood.Whereas the architecture of striated muscle myofibrils is relatively well understood, much less is known about the contractile actomyosin bundles of smooth muscle and non-muscle cells. Stress fibers are key mechanosensing elements in non-muscle cells, and they share many structural components with muscle sarcomeres13. The N-terminal ~200 residue region of Caldesmon interacts with both myosin II and calmodulin14, and the C-terminal ~200 residue region binds actin filaments, tropomyosin, and calmodulin18. The C-terminal region of Caldesmon inhibits the actin-activated ATPase of myosin II in vitro, and the binding of calmodulin to this region decreases its affinity for F-actin and releases the inhibition21. Based on biochemical studies, several different functions for Caldesmon were proposed. According to the popular view, Caldesmon regulates actomyosin contractility in a Ca2+/calmodulin and phosphorylation-dependent manner and is hence functionally equivalent to the skeletal muscle troponin complex13. Caldesmon was additionally proposed to stabilize actin filaments to maintain the structural integrity of actomyosin bundles23, and stabilize the latch-state of myosin II capable of maintaining force for long periods with low energy consumption24.Although stress fibers and smooth muscle actomyosin bundles lack certain key components of striated muscle myofibrils, they contain other proteins that may functionally replace central striated muscle components. These include actin/myosin-binding protein Caldesmon, which has smooth muscle  and non-muscle cell  specific splice variants. Apart from the ~250 residue regions in the center of H-Caldesmon, the two splice variants are identical to each other25, whereas specific deletion of the H-Caldesmon resulted in defects in arterial muscle relaxation26. In non-muscle cells, Caldesmon over-expression increases the thickness of stress fibers in lung carcinoma A549 cells, and depletion of Caldesmon by RNAi resulted in thinner stress fibers27. On the other hand, over-expression of Caldesmon decreased the contractility of SV80 cells28. Whether these phenotypes result from defects in actomyosin bundle organization, increased contractility of actomyosin bundles, from the loss of myosin II latch-state, or from some other defects in actomyosin bundles is not known. Altered expression levels of Caldesmon are also linked to the progression of many cancers30 and developmental abnormalities33, but the underlying mechanism has remained elusive.Despite extensive biochemical data, the in vivo function of Caldesmon has remained elusive. Knockout mice, lacking both L-Caldesmon and H-Caldesmon, die perinatallyHere, we revealed that Caldesmon is an extended protein that displays a highly dynamic association with stress fibers. Importantly, studies on Caldesmon knockout and rescue cells provide evidence that Caldesmon does not function as a negative regulator of myosin II activity, but that it instead cross-links myosin filaments and actin-tropomyosin filaments to maintain the regular spacing of myosin II filaments within actomyosin bundles. Our findings reveal how force imbalances in actomyosin bundles, and the organization of the mechanosensitive stress fiber networks, are controlled through dynamic cross-linking of actin and myosin filaments.34, but its precise localization within actomyosin bundles has not been reported. We applied 3D super-resolution structured illumination microscopy (3D-SIM) on U2OS cells immunostained with Caldesmon-specific antibody, and on cells expressing mCherry-fusion of Caldesmon. Both endogenous Caldesmon and mCherry-Caldesmon displayed periodic localization patterns along the contractile ventral stress fibers and transverse arcs but were absent from the non-contractile dorsal stress fibers  experiments on ventral stress fibers of U2OS cells expressing either GFP-Caldesmon or GFP-NM-IIA revealed that Caldesmon is a highly dynamic stress fiber component, with halftime of approximately 1–2 s. As reported earlier38, the recovery of NM-IIA heavy-chain was much slower compared to Caldesmon , which binds myosin II in vitrolls Fig.  displayells Fig. . Howeverlls Fig.  and displls Fig. . The exp40 and interaction of the full-length smooth muscle Caldesmon to actin filaments was reported to be enhanced by Tpm19, we examined the effects of different stress fiber-associated Tpms on actin interactions of Caldesmon. Actin filament co-sedimentation assay revealed that both full-length Caldesmon and its C-terminal region (residues 380–501) bind actin filaments, with the affinity of the full-length Caldesmon being slightly higher  on wild-type and Caldesmon knockout cells. Surprisingly, these experiments revealed that Caldesmon knockout cells exert 50–75% diminished contractile forces onto the substrate as compared to the wild-type U2OS cells  model to obtain a time constant (τ) and plateau retraction distance (Lo). In some cases, arc retraction did not fit KV retraction kinetics, perhaps because of the disconnect between the curved morphology of the arcs and the linear structure assumed by the KV model. We therefore subjected all SFs to a second, model-independent analysis in which we measured the distance retracted by one end of a cut fiber 60 s after ablation  phosphorylation was unaltered in the Caldesmon knockout cells  Caldesmon is an extended and highly dynamic component of contractile stress fibers, where it links myosin II filaments to specific tropomyosin-actin filaments, (2) Caldesmon does not function as a negative regulator of myosin II as previously proposed but instead maintains the regular spacing between individual myosin II filaments within stress fibers by preventing aberrant lateral ‘sliding’ of myosin filaments, and (3) Loss of Caldesmon leads to defects in the organization, contractility, and mechanosensing of the stress fiber networks, and to consequent problems in cell morphogenesis, migration, and invasion.52, as well as with biochemical work providing evidence that the C-terminal region of H-Caldesmon binds actin and tropomyosin, while the N-terminal region of the protein interacts with myosin II57. Interestingly, our results propose that Caldesmon associates slightly better with actin filaments decorated by non-muscle tropomyosin isoforms Tpm1.6, Tpm2.1, and Tpm3.2, as compared to Tpm4.2-decorated or bare actin filaments. In this context, it is important to note that stress fibers are composed of functionally distinct actin filaments that are specified by different tropomyosin isoforms, and from these Tpm4.2 is most closely associated with myosin II function58. Caldesmon may thus preferentially interact with those actin filaments that do not form ‘tracks’ for myosin II movement. These data, together with our FRAP and domain depletion experiments, demonstrate that Caldesmon is a dynamic component that cross-links myosin II to tropomyosin-actin filaments , is an extended molecule, whose N-terminal domain associates with the neck regions of myosin II molecules within NM-IIA filaments, while the C-terminal region of L-Caldesmon binds specific tropomyosin–actin filaments. These data are in agreement with previous electron microscopy work on purified smooth muscle H-Caldesmon demonstrating that it is an elongated molecule in vitronts Fig. .27. However, several lines of evidence from our work are not consistent with these earlier models. First, loss of Caldesmon results in decreased overall contractility and retrograde flow of the stress fiber network. Caldesmon depletion also does not increase the pre-stress within individual stress fibers, as would be expected if Caldesmon would function as a troponin-like negative regulator of myosin II activity. Second, the N-terminal myosin II-binding domain of Caldesmon is absolutely critical for the cellular function of the protein. On the other hand, the isolated C-terminal actin-binding region of Caldesmon still localizes to stress fibers, but cannot rescue the Caldesmon knockout phenotype, although previous biochemical studies proposed that this domain alone can regulate myosin II movement along actin filaments in a Calmodulin-regulated manner13. Finally, Caldesmon is a highly dynamic component of stress fibers and appears to preferentially associate with those tropomyosin–actin filaments that are not linked to myosin II movement. Thus, Caldesmon is not well-suited to function as a ‘road block’ for myosin II movement. It is also important to note that, since stress fibers are still present in Caldesmon-depleted cells, and because the N-terminal myosin II-binding domain is critical for its function in non-muscle cells, Caldesmon is also unlikely to function as an important stabilizer of actin filaments within stress fibers.Previous studies proposed that Caldesmon functions as a negative regulator of myosin II and/or it stabilizes actin filaments within contractile actomyosin bundles6. However, at the molecular level Caldesmon is very different from the M-band proteins, because Caldesmon links myosin II to tropomyosin–actin filaments, and is a highly dynamic protein within stress fibers. We speculate that this is due to the rapid dynamics of stress fibers compared to myofibrils. Thus, in stress fibers, there is a need for a more dynamic system that maintains uniform myosin II distribution and force balance. Our results, together with earlier studies63, also suggest that the organization of actin filaments must be different between skeletal muscle sarcomeres and stress fibers of non-muscle cells. Unlike the highly regular length of actin filaments in muscle sarcomeres, we propose that tropomyosin-actin filaments, especially in transverse arcs, are of irregular length and can often extend beyond the adjacent α-actinin-rich Z-disk-like regions of stress fibers. This would allow myosin II filaments to move long distances laterally along transverse arcs, as observed in our live-imaging experiments. Moreover, loss of Caldesmon appears to result in the aberrant organization of actin filaments within stress fibers, together with laterally moving myosin II filaments. Although F-actin and tropomyosin distributions appear quite regular within the stress fibers of Caldesmon knockout cells, α-actinin appears to form larger aggregates in the absence of Caldesmon, and the distributions of α-actinin and Tmod1 along stress fibers are less regular in Caldesmon knockout cells. We speculate that these phenotypes are linked to the aberrant lateral movement of myosin II filaments because there are no reports indicating that Caldesmon would interact with α-actinin or Tmods.Our results reveal that Caldesmon prevents abnormal lateral ‘sliding’ of myosin II filaments along actomyosin bundles, and is thus critical in maintaining uniform spacing between individual myosin II filaments within stress fibers Fig. . Thus, r20. Thus, the stress fiber localization of Caldesmon may be controlled by Ca2+/calmodulin. Moreover, as an elongated protein that links the necks of myosin II filaments to actin filaments, Caldesmon could, in principle, also function as a mechanosensor within stress fibers. However, our FRAP experiments revealed that Caldesmon is a highly dynamic component of stress fibers, and its dynamics are not affected by the inhibition of myosin. Thus, if Caldesmon also functions as a mechanosensor, it is most likely able to respond to forces only in a very short time scale.In addition to linking myosin II filaments to Tpm-actin filaments, Caldesmon may also have more specific functions in cells. This is because Caldesmon also binds calmodulin, and this interaction inhibits the actin-binding of Caldesmon13. Our experiments demonstrate that in osteosarcoma cells, Caldesmon depletion results in decreased cell migration and invasion. However, in the complex tissue environment the loss of Caldesmon, and hence the defects in force-balance of actomyosin bundles, are expected to have different effects on cancer progression, depending on the type of cancer and tissue environment. In this context, it is important to note that based on the nuclear localization of YAP, our experiments suggest Caldesmon knockout cells are unable to sense substrate stiffness which is a property associated with cancer progression64.In addition to its role in animal physiology, altered expression levels of Caldesmon were also linked to multiple cancers11. Briefly, cells were cultured in 4.5 g/L glucose containing DMEM , supplemented with 10% fetal bovine serum  and Pen-Strep-Glutamine solution  in a humidified atmosphere at 37 °C, 5% CO2 and 95% relative humidity. Cells were regularly tested for mycoplasma contamination using the Mycoalert™ Mycoplasma Detection Kit . For overexpression studies, FuGENE® HD transfection reagent  was used as per the manufacturer’s instructions with few standardizations using a 3.5:1 FuGENE HD/DNA ratio. Transfections were carried out for 24 h before live-cell imaging or before fixation with a 4% paraformaldehyde (PFA) solution. For rescue experiments, cells were transfected for 48 h.Human osteosarcoma (U2OS) cells  were maintained as previously describedPara-aminoblebbistatin  was used for the NM-IIA inhibition. Fibronectin  was used for surface-coating in live and fixed-cell imaging studies. Treated coverglass was mounted using ProLong™ Glass Antifade Mountant .The following antibodies were used for western blotting (WB) and immunofluorescence (IF) microscopy: 1:10,000 (WB), 1:400 (IF) rabbit anti-Caldesmon ; 1:500 mouse anti-Caldesmon ; 1:400 mouse anti-Vinculin ; 1:1000 (WB), 1:800 (IF) rabbit anti-NM-IIA ; 1:200 (WB) mouse anti-RLC ; 1:1000 rabbit anti-phospho-MLC  ; 1:500 rabbit anti-α-actinin1 ; 1:500 rabbit anti-YAP ; 1:5,000 rabbit anti-GAPDH ; 1:200 mouse anti-Tropomyosin 1 and 2 . 1:200 mouse anti-Tropomyosin 2 and 4 (LC24) and 1:200 mouse anti-tropomyosin 3 (γ−9d) antibodies were kind gifts from Peter W. Gunning . Alexa Fluor phalloidin 488 , phalloidin 568  and phalloidin 647  were used for filamentous-actin visualization; Alexa Fluor goat anti-mouse 488 , 568 , and 647 ; Alexa Fluor goat anti-rabbit 488  and 647  were purchased from Thermo Fisher Scientific for IF microscopy. Goat anti-rabbit HRP-conjugate (G21234), and goat anti-mouse HRP-conjugate  were purchased from Thermo Fisher Scientific for Western blotting experiments. 4’,6-diamidino-2-phenylindole, dihydrochloride  was used for nuclear staining. Pageruler Plus prestained protein ladder  was used as a molecular weight marker in SDS–PAGE. 34; mCherry-non-muscle Caldesmon was generated by amplifying Caldesmon-encoding fragment from rat non-muscle-Caldesmon construct. The fragment was sub-cloned further into pmCherry-C1 vector using the XhoI/BamHI restriction sites in the MCS region. For expression studies in U2OS cells, different domain deleted versions of Caldesmon namely—Cald(1-200), Cald(201-531), Cald(380-531), Cald(427-531), Cald(453-531), Cald(497-531), Cald(1-20), Cald(1-200/374-531), Cald(1-200/427-531), Cald(1-200/453-531), and Cald(1-200/497-531) were produced from mCherry-full-length Caldesmon by PCR, and sub-cloned into a relevant plasmid. For NM-IIA rescue experiments using smooth muscle-specific Caldesmon , pDONR221-Hs-H-Caldesmon (HsCD00746163) was procured from DNASU plasmid repository  and was sub-cloned further in pmCherry-C1 vector. For biochemistry, FL-Cald, Cald(1-200), Cald(201-531), and Cald(380-531) were tagged with His10pSUMO in pBR322 vector. All primers used for cloning purposes are listed in Supplementary Table 65. pSpCas9 (BB)−2A-GFP vector was a gift from F. Zhang (Addgene plasmid #48138). CMV-GFP-NMHC-IIA was a gift from Robert Adelstein (Addgene plasmid #1347)66 and NM-IIA-GFP was a gift from Matthew Krummel (Addgene plasmid #38297). GFP-lifeact was a kind gift from Maria Vartiainen  and pEGFP-C1-F-tractin was a gift from Dyche Mullins (Addgene plasmid #58473). eGFP-CP-β2 was a kind gift from D. Schafer 67. pEGFPN1-HsTmod1_wt was available from a previous study11.The following plasmids were used in this study: rat GFP-non-muscle-Caldesmon (gift from A. Bershadsky)71. Guide sequences 5’-CACCGGCCGTTCCTGTCGGGCTCGG and 5’-CACCGGGGACAGGTGACCGACCAGG specific to exon 1 of Caldesmon gene having highest on-target efficacy scores were selected based on CRISPR design tool and were cloned into pSpCas9 (BB)−2A-GFP vector. The constructs were transfected in U2OS cells and positive cells were sorted individually with FACSAria II cell sorter (BD) supplied with BD FACSDiVa Software v 8.0 into a 96-well plate containing DMEM media supplemented with 20% FBS and 25 mM HEPES. Caldesmon deletion in these knockout cell lines was confirmed on protein level by Western blotting, and the underlying genomic alteration was identified by Sanger Sequencing (Eurofins genomics sequencing service) and next generation sequencing (NGS) (Illumina MiSeq.) that was performed at the DNA Sequencing and Genomics Laboratory (BIDGEN) laboratory .Caldesmon knock-out cell lines were generated by CRISPR/Cas9 method as described previouslyE. coli BL-21 (DE3) pLys-S cells and grown in 2X lysogeny broth containing Kanamycin (25 µg/ml) and Chloramphenicol (35 µg/ml) at 37 °C until the OD600 reached 0.8–1.0. Protein expression was induced with 0.5 mM IPTG for 24 h at R.T. Cells were harvested by centrifugation , re-suspended in Ni-binding buffer  and lysed thoroughly by sonication. Cell lysates were mixed with Ni-NTA agarose beads  in a Nickel binding buffer at 4 °C for 1 h. The beads were washed with four column volumes (CV) of binding buffer, 6 CV of 1 M NaCl solution followed by elution with Ni-elution buffer . SUMO tag was removed by SENP protease overnight in dialysis buffer  and proteins were further purified on gel filtration column pre-equilibrated with 20 mM HEPES pH-8.0, 50 mM NaCl. Fractions containing Caldesmon proteins were pooled together, concentrated, loaded in ion exchange HiTrap Q FF columns , washed with 10 mM Tris–HCl pH 7.5, 10 mM NaCl, and eluted using 10 mM–1 M NaCl salt gradient. Finally, the fractions containing pure protein were pooled together, concentrated with Amicon® Ultra-15 centrifugal filters , and protein concentration measured with nanodrop using an extinction coefficient of (protparam), snap frozen in liquid nitrogen and stored at −80 °C.Full-length Caldesmon and its truncated versions cloned in pBR322 were expressed in 65. Non-tagged tropomyosins were purified as per a published method72 with some essential modifications. Briefly, non-tagged tropomyosins were expressed in E.coli BL21-DE3 cells in 1 l LB broth (2X) containing ampicillin (100 µg/ml) and were grown until the O.D600 reached 0.8–1.0. The protein expression was induced with 1 mM IPTG at 37 °C for 3 h. The induced cells were harvested by centrifugation, resuspended in lysis buffer , and were lysed thoroughly by sonicating  until the bacterial suspension turns sufficiently transparent. Further, the cell lysate was heated in a water bath set at 80 °C for 8 min. The heat-inactivated cell lysates were subsequently cooled to room temperature in a water bath. The supernatant containing Tpms was cleared from denatured proteins and cell debris by centrifugation at 47,810×g, 4 °C for 45 min. The supernatant was collected in a fresh tube and the Tpms were acid precipitated to pH 4.7 by adding drop-by-drop 2 M HCl while mixing. The precipitate containing Tpms was centrifuged at 3900×g, 4 °C for 12 min, and the pellet was resuspended in resuspension buffer . In order to resuspend the pellet completely, the buffer pH was re-adjusted to 7.0 using 1 M NaOH. The acid precipitation was repeated two more times to remove impurities and obtain a white pellet. The white pellet was re-suspended in pellet resuspension buffer and was filtered using a 0.22 µm membrane filter. Dialysis was performed overnight in dialysis buffer  using 10 kDa molecular cutoff membrane . The next day, a 4 ml SP QFF anion exchange column was equilibrated with 4 column volumes of anion exchange wash buffer  and the supernatant was loaded onto the column at speed of 2 ml/min. The column was washed with four column volumes of wash buffer. The column was further equilibrated with anion exchange elution buffer  and Tpm was collected in the flow through. Samples from peak fractions were analyzed by SDS–PAGE and fractions were pooled together. The pooled fractions were diluted four times with hydroxyapatite wash buffer  and loaded on an equilibrated hydroxyapatite column at 0.5 ml/min speed. The column was washed with four column volumes of hydroxyapatite wash buffer and the protein was eluted in a 10–240 mM phosphate gradient against hydroxyapatite elution buffer . The eluted fractions were analyzed by SDS–PAGE and the fractions containing Tpms were pooled together. As the elution volume was large, we utilized an acidification procedure to concentrate the protein. The protein pellet was resuspended in dialysis buffer , protein concentration was determined with nanodrop, the protein sample was aliquoted, flash-frozen, and stored at −80 °C.The plasmids for the structural and biochemical studies of non-tagged human tropomyosins Tpm1.6, 1.7, 2.1, 3.1, 3.2, and 4.2 were from earlier study6 cells were plated on 35 mm dishes 18–24 h before harvesting. Cells were washed twice with ice-cold PBS solution and the cells were harvested on ice in PBS containing protease and phosphatase inhibitor cocktails using a cell scrapper. Pelleted cells were lysed using 4× Laemmli buffer + β-ME. Whole-cell extracts were run on 4–20% gradient SDS–PAGE gels  followed by transfer onto 0.2 µm nitrocellulose membrane  using Trans-Blot® Turbo™ transfer system . Blots were blocked for 1 h at RT in 2.5% BSA in PBS-T (0.05% Tween-20) followed by probing with primary antibody against phospho- and total-proteins on overnight shaking at 4 °C. The blots were washed three times in 0.05% TBS-T and incubated with respective HRP-conjugate secondary antibodies for 1 h at RT. Finally, the blots were washed and protein bands were detected using WesternBright™ ECL-spray  in the ChemiDoc XRS+ System . Protein bands were quantified using Image Lab™  and Fiji ImageJ 1.53c [https://imagej.nih.gov/ij/] software. GAPDH was used to normalize expressions of other proteins and phospho-expression was normalized with respective total-protein levels. Densitometry analysis of Western blots and the uncropped Western blots are provided in the Source Data files.A total of 2 × 1073 with few modifications. Non-muscle actin (β/γ-actin from human platelets) was purchased from Cytoskeleton Inc. and used as per the manufacturer’s instructions. Different amounts of either β/γ-actin or β/γ-actin and non-tagged tropomyosin 1.6, 2.1, 3.2, and 4.2 were mixed in presence of G-buffer . In order to saturate actin with tropomyosins, we mixed actin and tropomyosins in 4:1 ratio. Caldesmon concentration was maintained at 1 µM for all experiments. Incubations were done at RT. Polymerization was carried out for 30 min using F-buffer . 1 µM of Caldesmon was added to the polymerized actin/actin: Tpm mix and was incubated for 30 min. The complex was centrifuged at 157,000×g for 60 min at 10 °C . Supernatant and pellet fractions were prepared for SDS–PAGE analysis by adding Laemmli buffer. The protein bands were separated on 4–20% gradient SDS–PAGE gels  and stained using 1% Coomassie staining solution  . The intensities of Caldesmon bands were quantified with QuantityOne program (Bio-Rad). The graph was plotted as the concentration of Caldesmon in pellet (μM) on Y-axis vs. actin/actin:Tpm1.6/actin:Tpm3.2 (μM) on X-axis.We performed actin co-sedimentation assays as previously reported74. Briefly, cells were seeded on 10 mg/ml fibronectin-coated coverglass and allowed to spread for 6 h. The old media containing floating cells was removed and coverslips were washed twice with PBS. Cells were fixed using 4% paraformaldehyde in PBS for 15 min at RT, followed by three washes with PBS-T (0.02% Tween-20). Cells were permeabilized with 0.1% Triton X-100 in PBS  and blocked with 1% BSA in a humidified chamber for 1 h at RT. Cells were immunostained with primary antibodies for 1 h at RT, washed three times with PBS-T, and incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at RT in dark. When relevant, F-actin and nuclei were visualized using phalloidin and DAPI. Finally, the coverslips were washed with PBS-T three times, with PBS once, and mounted using an anti-fade mounting reagent.Immunostaining (both for wide-field and 3D-SIM) was performed as reported previouslyStained cells were visualized at RT on a Leica DM5000B and DM6000B upright microscope with ×63/1.40-0.60 HCX PL APO oil objective, equipped with Semrock bright line DAPI, FITC/GFP, mCherry/TRITC, and Cy5 filters. Wide-field fluorescence images were captured using Hamamatsu Orca-Flash4.0 V2 sCMOS camera with image resolution 2048 × 2048 pixels. The images were acquired using LAS X 3.4.2 software and processed using Fiji ImageJ 1.53c software.Wild-type and Caldesmon KO cells were seeded on Softview™ 35 (10 mm Glass Bottom), Easy Coat™ dishes of 8 kPa  and 50 kPa (SV3520-EC-50 PK) substrate stiffness. Confocal imaging was performed at RT on Andor Dragonfly confocal microscope using ×10 Plan Fluor DL/0.3 NA/15.2 WD and ×20 S Plan Fluor ELWD/0.45 NA/8.2–6.9 WD objectives, equipped with 445/521/594/685/809 filters for imaging. Images were captured using Andor iXon 888 U3 EMCCD camera and visualized using Fusion 2.0 iXon SRRF-Stream real-time super-resolution software.37. GE immersion oil calculator was used to calculate the refractive index of oil to be used while imaging images stained with two or more fluorophores. SIM images were constructed from average intensity projections of Z-stacks using Stacks/Z-project and analyzed using 3DimageJ plugins in imageJ 1.53c software. For the line-scan analyses, mCherry-Caldesmon doublets (with a distance of ~0.2 μm between the two peaks) were chosen and manually centered on the analyzed ROI. The fluorescence intensities of mCherry-Caldesmon and the corresponding second protein were then measured along the ROI.For SIM imaging, precision coverslips with 170 µm thickness were used. The imaging was performed at RT on a GE Deltavision OMX super-resolution upright microscope with a Plan-Apochromat N ×60/1.42 Oil immersion objective, equipped with 405, 488, 568, 640 nm diode lasers and sCMOS detector camera. Images were acquired using AquireSR 4.4 software, reconstructed, and aligned using SoftWoRx 7.0 software as described beforeFor the analysis of cell shape and size, wild-type and Caldesmon KO untransfected/mCherry-Caldesmon transfected U2OS cells were plated in a fibronectin-coated 96-well plate  and incubated for 6 h. The plated cells were counter-stained with DAPI, and phalloidin 488. Cell imaging was done at RT using ImageXpress® Nano Automated Imaging System (Molecular Devices) with ×20/0.45S Plan Fluor ELWD, WD 8.2–6.9 mm water immersion objective, and 409, 495, 593, and 660 nm dichroic filters. The images were processed in CellProfiler 3.1.8 automated image analysis software. A two-color and three-color pipeline was designed for machine learning and to process untransfected wild-type, Cald KO, and transfected Cald KO U2OS cells. DAPI served as a primary object filter for cell segmentation. Object aggregates and smaller artifacts were discarded automatically based on intensities by setting pre-filters like threshold and correction factor. After the primary screening, secondary objects positive for phalloidin and GFP-Caldesmon were identified. Post-regularization, the intensities of objects were measured and filtered and the shape-size of the positive objects was calculated automatically.42. Wild-type and Caldesmon KO U2OS cells expressing RFP-actin and NM-IIA-GFP constructs were plated on fibronectin-coated 35 mm glass bottom dishes  after 24 h of transfection. The cells were incubated for attachment for 2 h. Time-lapse live-cell imaging of RFP-actin and NM-IIA-GFP myosin motor accumulation and retrograde flow was performed using Zeiss LSM 880 upright confocal microscope combined with AiryScan (Fast) detector at 37 °C, with a Plan-Apochromat ×63/1.4 NA/0.19 WD Oil immersion objective. Time-lapse images were captured using Zeiss Zen 2.0 software for 30 min at 10 s intervals. Deconvolution was done using ZEN black software. The retrograde flow of NM-IIA in wild-type and Cald KO cells was manually analyzed in Fiji imageJ 1.53c. Wild-type and Caldesmon KO U2OS cells were transfected with GFP-tractin for 24 h, and re-plated before imaging for 2 h on fibronectin pre-coated CYTOOchips™ circular micropatterns 35 mm coverglass  attached with a CYTOOchamber™ . The micropatterns were washed twice with pre-warmed PBS and time-lapse images were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations)—a fully motorized Zeiss inverted spinning disk confocal microscope (Zeiss Axio Observer Z1). The microscope is equipped with WC-Apochromat ×63/1.2 NA/Corr. WD = 0.28 M27 water objective and Yokogawa CSU-X1 M1 5000 rpm confocal scanner with Andor Neo sCMOS camera. The microscope has a 405/488/561/640 dichroic filter and a brick CO2 mixer with active temperature and humidity control. Images were acquired with Slidebook 6.0.15 software. The flow of transverse arcs in wild-type and Caldesmon KO U2OS cells were tracked manually in Fiji imageJ1.53c software and the velocity of retrograde flow of transverse arcs was calculated.Live cell imaging was performed as previously reported42. To measure the dynamics of actin, Caldesmon, and NM-IIA, wild-type, and Caldesmon KO U2OS cells were transfected with GFP-actin, GFP-NM-IIA, and GFP-Caldesmon constructs, incubated for 24 h before seeding them on fibronectin-coated glass-bottomed 35 mm imaging dishes  for 2 h. The old media containing un-attached cells was replaced with fresh media before live-cell imaging on 3I Marianas imaging system (3I Intelligent Imaging Innovations)-Yokogawa CSU-X1 M1 5000 rpm spinning disk confocal microscope at 37 °C and CO2 control using ×63/1.2 W C-Apochromat Corr WD = 0.28 M27 water objective and Andor Neo sCMOS camera. A bleaching area was marked on ventral stress fibers having uniform length and thickness across different transfected groups. Before bleach (100% intensity with 150 mW–488 nm laser power for 1 × 1 ms), five pre-bleach images were captured, followed by bleaching and post-bleach imaging to monitor photo-recovery at 50 × 200 ms and then at 300 × 1 s, respectively. To measure the photo-recovery, the intensity of the bleached area was normalized to the neighboring non-bleached area on the same stress fiber. Normalized values of different groups obtained from experimental repeats were used to calculate the mean recovery and variation across replicates for actin, Caldesmon, and NM-IIA in wild-type and Caldesmon KO U2OS cells.FRAP experiments were performed as described previously76 on glass-bottomed 35 mm imaging dishes . The gels were covered with fluorescent 488-nano beads (diameter 200 nm) and after that further coated with collagen-1. Prior to cell plating, gels were incubated with warmed 10% FBS and supplement-containing DMEM media for 1 h.The polyacrylamide (PAA) gel substrates with 26 kPa stiffness (Young’s modulus) were prepared as described previously78. Cell borders were manually tracked and analyses of different groups were performed as blind analyses.Wild-type and Caldesmon KO U2OS cells transfected with GFP-Lifeact construct were incubated for 24 h. Wild-type, untransfected and mCherry-FL-Caldesmon transfected U2OS cells were cultured on the collagen-1-coated 35 mm imaging dishes for 2 h at 37 °C. After a brief wash, we imaged the attached cells and the fluorescent beads beneath the cells using the 3I Marianas imaging system (3I Intelligent Imaging Innovations) by bright field and epi-fluorescence settings before and after cell-detachments by trypsinization (microscope specifications and imaging setup as described before in live-cell imaging section). Displacement maps were received by comparing the bead images before and after trypsinization. Traction fields were computed from the displacement data by utilizing the principle of Fourier transform traction cytometry as described before48. Briefly, U2OS cells were transduced with RFP-Lifeact and cultured on either unpatterned surfaces, rectangular frame patterns, or crossbow patterns. Micropatterns were made as previously described48 Briefly, coverslips were plasma-treated and then incubated with 50 µg/ml poly-l-lysine conjugated to polyethylene glycol  in 10 mM HEPES, pH 7.4, for 1 h at room temperature, then rinsed with PBS and deionized water. To pattern the surface, coverslips were placed under a quartz-chrome photomask with micropattern features . The mask and coverslips were then illuminated using 180 nm UV light (Jelight) for 15 min and then rinsed with PBS. Both patterned and unpatterned coverslips were incubated with 20 µg/ml fibronectin (EMD Millipore) in 100 mM bicarbonate solution, pH 8.5, overnight at 4 °C, then rinsed with PBS. U20S cells were seeded 3000–5000 cells/cm2 and incubated for 4–6 h at 37 °C to allow for attachment and spreading. Imaging and ablation were performed with a Zeiss LSM 780 confocal microscope system equipped with a Spectra-Physics Mai-tai laser for multiphoton imaging and ablation. Single stress fibers were then photo-irradiated with the femtosecond laser to induce an incision. The retraction was tracked every 1 s for 75 s and fit to a Kelvin–Voigt single-exponential model to extract a viscoelastic time constant (τ) and plateau retraction distance (L0). Retraction curves were excluded from the τ and L0 datasets if they did not meet all of the following criteria for the fitted KV equation: (1) adjusted R2 > 0.9, 2) τ  < 0.8 * imaging window (τ < 60), and (3) sum squared errors of prediction < 10. The model-independent analysis of retraction was calculated by subtracting the distance between the severed ends at 60 s and at 0 s and dividing by 2.Laser ablation studies were performed as previously described70. Wild-type and Caldesmon KO cells untransfected or GFP-FL-Caldesmon transfected were cultured for 2 h on a 12-well plate coated with fibronectin before imaging. Live-cell imaging was performed using a Cell-IQ imaging system by Nikon Plan Fluor ×10/0.30 Ph1 DL objective (fluorescent and phase-contrast mode) and the time-lapse images of cells were captured with Qimaging Retiga EXi camera (pixel no. (W × H): 1392 × 1040) with 2 × 2 binning at 10 min intervals for 24 h. The system utilizes Cell-IQ Imagen 4.1.0 and Cell-IQ Analyser 4.4.0 software for cell imaging and analysis. The fluorescence and phase-contrast images were stitched and combined together to differentiate between transfected and non-transfected cells. To assess the migration velocity in Caldesmon KO-rescue cells, only GFP-Caldesmon-transfected cells were analyzed, while non-transfected cells determined the Caldesmon KO cell migration velocity. Cells colliding with each other or undergoing division were omitted during the analysis and the measured migration velocities across groups were used to plot scatter box plots.Random cell migration velocities were studied as reported previously5 cells were seeded in a 12-well culture plate and incubated for 18–24 h to form a uniform cell-monolayer. Scratch was made with 200 µl micropipette tip in the mid-area of every well, media replaced with 10% FBS + 10 mM HEPES containing-DMEM media and time-lapse live cell imaging performed using Cell-IQ imaging system by Nikon Plan Fluor 10x/0.30 Ph1 DL objective (phase-contrast mode). Images were captured with Q imaging Retiga EXi camera (pixel no. (W × H): 1392 × 1040) with 2 × 2 binning at 10 min intervals for 24 h using Cell-IQ Imagen 4.1.0software. Time-lapse images were stitched, analyzed to obtain wound area covered (%), length covered (µm) calculated with Cell-IQ Analyser 4.4.0 software, and used to plot a line graph as means of percent area covered by U2OS wild-type and Caldesmon KO cells from three individual repeats.Wound-healing property of wild-type and Caldesmon KO U2OS cells was assessed by wound healing assay. 5 × 1079. Cell invasion was performed using 6.5 mm with 8.0 μm pore polycarbonate membrane insert  as per manufacturer’s guidelines. 500 µl of 300 µg/ml matrigel was added into the transwell insert without creating bubbles and allowed to polymerize for 30 min at 37 °C without letting it dry. 500 µl of serum-free DMEM media was added to the upper chamber of the insert while 20% FBS containing DMEM was added to the lower chamber, i.e., into the well such that the insert was immersed into the media. 25 ng/ml epidermal growth factor  was added to the lower chamber as a chemoattractant. U2OS wild-type and Caldesmon KO cells were trypsinized, suspended in 1.5% FBS containing DMEM media, added 5 × 104 cells to the upper chamber, and incubated for 6 h at 37 °C. Following incubation, the inserts were removed from wells, washed with PBS, scrapped with moist cotton swabs from inside the insert to remove cells, fixed , permeabilized (0.1% Triton X-100), and counterstained with DAPI and phalloidin 488 dyes. The insert was cut, mounted on a slide on Vectashield® Vibrance™ antifade mounting medium , and images were captured with Leica DM5000B upright microscope equipped with ×20/0.7 HC PL APO CS wd = 0.59 objective, DAPI-5060C  and GFP-4050B  filters and Hamamatsu Orca-Flash4.0 V2 sCMOS camera. Minimum five images across different view-fields captured for wild-type and Caldesmon KO cells from three replicates. The images were visualized using LAS X 3.4.2 and processed using Fiji imageJ 1.53c software.The 3D-matrigel assay was used to assess the invasive property of wild-type and Caldesmon KO U2OS cells as reported earlierTo analyze the fluctuations in myosin filaments in wild-type and Caldesmon-depleted U2OS cells, myosin filaments in transverse arcs flowing from the cell periphery towards the cell center were tracked for 30 min. Time-lapse images of selected transverse arcs collected at 100 s intervals were applied to draw the kymographs.80. 3D-imageJ suite is essential for executing DiAna tool. For DiAna colocalization analysis, GFP- and mCherry-tagged protein images were captured using GE Deltavision OMX super-resolution microscope as described in the super-resolution imaging section. SIM images constructed from average intensity projections of Z-stacks were used as source images in DiAna plugin. Green and red channel images were split, and segmented with the 3D-iterative segmentation tool in imageJ using the following criteria; min vol. pixels: 10, max vol. pixels: 100, min threshold/contrast: 10, elongation criteria (for F-actin), step threshold method with best segmentation and method value: 100. The segmented images were further uploaded in DiAna “analyze” option. % volume colocalization and distance between one or more red and green dots were measured from center-to-center, center-to-edge, and edge-to-center of red and green dots. The mean volume co-localized (%) and distance (µm) between two protein molecules in different groups were calculated and plotted. The complete tutorial is available online: [https://docs.google.com/presentation/d/e/2PACX-1vTcw7Zsc8gX4TsU1pwMsRlSVwVOa89sUQaGEwxWeANZsA3DCEwCU2PSvKruTVoN6V5D3DaDnNHI4-Yx/pub?start=false&loop=false&delayms=3000&slide=id.p].To determine the percent co-localization volume and distance analysis between two protein molecules in a three-dimensional space, Fiji imageJ 1.53c-based DiAna was utilized as described previously81. For analyzing the YAP localization in U2OS wild-type and Caldesmon KO cells . The graphs indicate the p-values for respective analyses.Single-blind analysis of different groups was performed for the following experiments: Traction force microscopy Fig. ; live-cells Fig. , YAP expFurther information on research design is available in the Supplementary informationPeer Review FileDescription of additional Supplementary FileSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Reporting Summary