The number of banana plants is closely related to banana yield. The diameter and height of the pseudo-stem are important morphological parameters of banana plants, which can reflect the growth status and vitality. To address the problems of high labor intensity and subjectivity in traditional measurement methods, a fast measurement method for banana plant count, pseudo-stem diameter, and height based on terrestrial laser scanning (TLS) was proposed.First, during the nutritional growth period of banana, three-dimensional (3D) point cloud data of two measured fields were obtained by TLS. Second, the point cloud data was preprocessed. And the single plant segmentation of the canopy closed banana plant point cloud was realized furtherly. Finally, the number of banana plants was obtained by counting the number of pseudo-stems, and the diameter of pseudo-stems was measured using a cylindrical segmentation algorithm. A sliding window recognition method was proposed to determine the junction position between leaves and pseudo-stems, and the height of the pseudo-stems was measured. Compared with the measured value of artificial point cloud, when counting the number of banana plants, the precision,recall and percentage error of field 1 were 93.51%, 94.02%, and 0.54% respectively; the precision,recall and percentage error of field 2 were 96.34%, 92.00%, and 4.5% respectively; In the measurement of pseudo-stem diameter and height of banana, the root mean square error (RMSE) of pseudo-stem diameter and height of banana plant in field 1 were 0.38 cm and 0.2014 m respectively, and the mean absolute percentage error (MAPE) were 1.30% and 5.11% respectively; the RMSE of pseudo-stem diameter and height of banana plant in field 2 were 0.39 cm and 0.2788 m respectively, and the MAPE were 1.04% and 9.40% respectively.The results show that the method proposed in this paper is suitable for the field measurement of banana count, pseudo-stem diameter, and height and can provide a fast field measurement method for banana plantation management. Banana is the main cash crop in tropical and subtropical regions. The banana industry is an important pillar for rural economic prosperity and farmers’ poverty alleviation in many areas of southern China , 2. The Karami et al.  used an The point cloud data of crops obtained by 3D reconstruction can be used to extract high-precision morphological and phenotypic parameters , 27. SonThere were the following problems in using TLS to count banana plants and measure pseudo-stem diameter and height: bamboo poles supporting banana plants and broken leaves would interfere with banana plant counting; how to segment single banana plants from banana populations due to closed canopy and serious cross shielding of leaves; and the measurement of pseudo-stem height needs to identify the junction between the lowest leaf and pseudo-stem and the junction between the ground and pseudo-stem.To solve the above problems, TLS was used to collect banana point cloud data in the nutrition growth period, and banana plant counting, pseudo-stem diameter, and height measurements were carried out. The number of plants was extracted from the fixed height point cloud data, and then a single plant was segmented using a combination of Euclidean clustering and K-means clustering. Finally, a method for calculating the pseudo-stem diameter and height of the banana pseudo-stem was proposed.The overview of the system is illustrated in Fig. The data collection experiment dates were April 14 and April 17, 2021. The study area is part of the crop planting base of Guangxi Jiejiarun Technology Co., Ltd. (GJTCL), which is an area of approximately 140 ha located in Fusui County, Chongzuo City, Guangxi Zhuang Autonomous Region, China. This area lies between the lat. 22°29′34.58″N and lat. 22°30′48.32″N and long. 107°46′7.03″E and long. 107°47′19.70″E. The banana cultivar was Williams B6, and its growth process can be roughly divided into four phenological periods: sucker, nutritional growth, bud bursting, and fruit maturation periods. During the experiment, bananas were in the late stage of the nutritional growth period, and the canopy was closed.2, and field 2 (F2) was 45 m long and 35 m wide, with a field area of 1575 m2. Prior to the experiment, the target balls were placed in the collection point cloud data area. Each site cloud data and other site cloud data were required to include at least three common target balls, and the target balls were not collinear. Trimble tx8 was installed at the base of the tripod. The scanning density level was set to level 2 and the scanning time was 3 min. The three leveling screws under the equipment were adjusted to ensure that the equipment was perpendicular to the horizontal plane. The scan key was clicked to start the station scan. The scanning stations were evenly distributed according to the scanning area, and point cloud data of the six stations were obtained in each field. The total scanning time was 18 min for each field, including the time to move the TLS, the total time to acquire the point cloud per field was about 30 min. The positions of the scanning site on the two Fields are shown in Fig. The Trimble tx8 TLS of the American Trimble company was selected to collect 3D point cloud data during the banana nutrition growth period; its performance parameters are listed in Table The Trimble Realworks software was used as a 3D point cloud data processing tool to extract and match the point cloud data. With Visual Studio 2013 as the platform, Point Cloud Library 1.8.0 (PCL1.8.0) and Cmake3.8.0, were installed. To measure the number of banana plants, pseudo-stem diameter, and height based on the point cloud data in the field, C +  + was used for software programming. The processing of 3D point cloud data includes three parts: data preprocessing, point cloud segmentation, and banana phenotypic parameter measurement. An overall point-cloud data-processing block diagram is shown in Fig. The data preprocessing procedure includes the following steps: point cloud data registration, background point cloud data removal, data downsampling, and format conversion. The banana point cloud data obtained from the field experiment were read using the Trimble Realworks software. The survey site cloud was established, and the point cloud data were registered using the target ball registration method. Through pass filtering, the background point cloud data were removed and the point cloud data of banana in the experimental area was extracted, F1, as shown in Fig. Banana point cloud segmentation includes five steps: segmentation of banana plants and ground point cloud data, removal of outliers, extraction of fixed-height point cloud, pseudo-stem segmentation, and single plant segmentation. Taking F1 as an example, a point cloud segmentation process was introduced. The processing process is illustrated in Fig. Step 1The preprocessed original point cloud is illustrated in Fig. Step 2The segmented plant point cloud data were statistically filtered to remove noise points in the banana plants. The results after removal are shown in Fig. Step 3 The point cloud data within  from the plane were extracted. For better Euclidean clustering, the extracted fixed-height point cloud data were statistically filtered to remove noise points around the pseudo-stem. The result is shown in Fig. Step 4The reserved point cloud was Euclidean clustering. The distances in the X-and Y-axes of each cluster of point cloud and their sum were calculated. The threshold was set to segment the pseudo-stem of a single banana plant from bamboo poles and broken leaves. The results are shown in Fig. Step 5 The number of segmented plant pseudo-stems was used as the K value in K-means clustering. The center point of the fixed-height pseudo-stem point cloud was used as the initialization center point for K-means clustering. The point clouds of the banana plants were clustered. The segmentation results are shown in Fig. Based on the segmented fixed-height pseudo-stem and single-plant point cloud, the following methods were used to automatically measure the number of banana plants, pseudo-stem diameter, and pseudo-stem height within the field: euclidean clustering, random sampling consistency cylinder segmentation, maximum traversal, and leaf pseudo-stem junction recognition. The specific measurement methods for the phenotyping parameters were as follows:The number of banana plants was calculated by counting the number of banana pseudo-stems using Euclidean clustering.To achieve high measurement accuracy, bamboo poles and leaves should be removed first. The distance sum in the X- and Y- axes of the bamboo pole point cloud was less than that of the banana pseudo-stem, and the distance sum in the X- and Y- axes of leaves point cloud greater than that of the banana pseudo-stem; therefore, the bamboo poles and leaves could be removed by setting the bounding box threshold, and the distance between the X- and Y- axes of the cluster point cloud was banana pseudo-stem within . The results are shown in Fig. The measurement of the pseudo-stem diameter of a banana includes two parts: the extraction of a fixed height point cloud of a single banana and the measurement of the pseudo-stem diameter.Fixed-height point cloud data of a single banana were extracted. The pseudo code of the method is presented in Method. 2 .Precision (P), recall (R), and percentage error (PE) were used to quantify the performance of the banana plant counting algorithm. The accuracy of the designed method was evaluated by comparing the automatic measurement of false stem diameter and stem height with the measurement of the artificial point cloud. The measurement accuracy was evaluated by the mean absolute percentage error (MAPE), root mean square error (RMSE), and determination coefficient RThe distance threshold of Euclidean clustering has an important impact on the classification of banana pseudo-stems, bamboo poles, and leaves. If the threshold is set too small, a leaf will be divided into multiple classes, resulting in a distance between the X- and Y- axes and within the threshold range of the pseudo-stem. The point cloud at the edge of the pseudo-stem is filtered out, resulting in a smaller distance and in the direction of the X-and Y-axes, which will be misidentified as a bamboo pole. If the threshold value is set too large, the leaves, pseudo-stems, bamboo poles, and pseudo-stems will be divided into one class, resulting in an increase in the distance between the X- and Y- axes, which will be misidentified as leaves. When the distance threshold is set too large or too small, the counting error of banana plant number will increase; therefore, it is necessary to set an appropriate distance threshold. The distance thresholds were set as 0.05 m, 0.10 m and 0.15 m respectively. The recognition results of the banana plant algorithm are shown in Fig. Under different distance threshold settings, the number of banana plants recognized by the algorithm and the number correctly recognized by the algorithm were counted. The calculation and evaluation indices used to measure the accuracy of the banana counting algorithm are shown in Table The pseudo-stem diameter of the point cloud after K-means classification was measured. A total of 187 banana plants were detected in F1, and 175 banana diameters were measured by cylindrical segmentation, of which only two were mistakenly identified as plants and 11 banana plants were not detected. In F2, 192 bananas were detected, and 177 banana diameters were measured by cylindrical segmentation, of which only one was mistakenly identified as a plant by the leaf. A total of 176 plants were correctly measured, and 24 banana plants were not detected. The main reason why the existing plants were not detected was that point cloud occlusion led to the loss of a fixed-height pseudo-stem point cloud, resulting in a smaller bounding box. Alternatively, the pseudo-stem was close to the broken leaf, resulting in the enlargement of the bounding box beyond its threshold range of the bounding box.2 was more than 0.95; the RMSE was controlled within 0.40 cm; the MAPE is not more than 1.30%, and the accuracy of automatic measurement of pseudo-stem diameter is not less than 98.70%. The results showed that this method had high accuracy in measuring the banana pseudo-stem diameter, and the measured value of the algorithm was consistent with the true value measured by the artificial point cloud.The banana pseudo-stem diameters of F1 and F2 were automatically and correctly measured using the program, and the results were compared with the artificial point cloud measurement results, as shown in Fig. After cylinder segmentation, the point cloud was recognized by the sliding window, the pseudo-stem height was measured, and the pseudo-stem heights of 175 bananas were measured in F1. Only two of these were mistakenly identified as plants by the leaves. In F2, the stem height measurement, only 1 of 177 bananas was mistakenly identified as plants by leaves, two pseudo-stems were measured for more than 5 m, and 174 plants were measured correctly. The pseudo-stem height measurement results exceeded the threshold mainly because point cloud occlusion led to the loss of banana leaf point clouds, and the contact position between the leaf and banana pseudo-stem could not be detected.2 was more than 0.46; the main reason was that the bamboo pole supported the banana plant, resulting in the low measured value of some pseudo-stem heights, and the banana pseudo-stem height was concentrated at 2.2 m, and the measurement error had a great impact on R2. The RMSE was controlled within 0.28 m; the MAPE was not greater than 7.60%, and the accuracy of program automatic measurement of pseudo-stem diameter was not less than 92.40%. The results showed that this method had a high accuracy in measuring banana pseudo-stem height, and the measured value of the algorithm was consistent with the true value measured by the artificial point cloud.The banana pseudo-stem heights of F1 and F2 were automatically and correctly measured using the program, and the results were compared with the artificial point cloud measurement results, as shown in Fig. 2 and F2 covers an area of 1575 m2. The phenotypic measurement results showed that F1 was better than F2 in terms of the measurement accuracy of plant number count, pseudo-stem diameter, and height. The main reason is that the area of F2 is larger than that of F1, and the density of the point cloud of F2 is lower than that of F1, which results in the missing of the petiole point cloud of some banana plants, and the measured value was too large. The point cloud is segmented into banana plants and bamboo poles, and the number of scanning stations is reasonably set, which can improve the accuracy of pseudo-stem height measurement.It can be seen from the automatic measurement results that the measurement accuracy of the pseudo-stem diameter is higher than that of the pseudo-stem height. The main reason is that, in the measurement of pseudo-stem diameter, threshold segmentation was used to extract fixed high pseudo-stem point cloud data, remove the interference of bamboo poles and leaves, and ensure the accuracy of cylindrical fitting. In the measurement of pseudo-stem height, there was no algorithm design for banana plants supported by bamboo poles, which leads to serious interference of bamboo poles on the measurement of pseudo-stem height of some banana plants. F1 covers an area of 931 mThe purpose of this study is to use TLS to collect high precision 3D point cloud data of banana plants in the fields, and to realize the automatic and accurate measurement of the number of banana plants, and pseudo-stem diameter and height.Compared to manual measurement, six scan stations were arranged for each field. The time of one scan is 3 min, the total scanning time is 18 min, considering the time of moving the TLS, the total time to acquire the point cloud per field is about 30 min. It takes an average of 3 min to manually measure the pseudo-stem height and diameter of a banana plant. There are 184 banana plants in F1, and the total time is 552 min. There are 200 banana plants in F1, and the total time is 600 min. Manual measurement takes approximately 20 times than TLS.Compared with TLS maize plant counting, Lin et al.  realizedCompared with Kinect V2 banana pseudo-stem diameter and height measurement, The MAPEs for diameter measurement were 1.34% and 2.34% , 29. In However, in pseudo-stem segmentation, the existing segmentation methods should be optimized to improve the segmentation accuracy; the method of segmenting bamboo and banana point clouds from point clouds should also be added to provide better point cloud data for subsequent phenotypic measurement. In the part of phenotypic parameter measurement, because there was a common value range between bamboo pole diameter and banana pseudo-stem diameter, there will be errors in banana plant counting using this method, which will also interfere with the measurement accuracy of pseudo-stem height. Therefore, it is necessary to further optimize the phenotypic parameter measurement methods. In addition, the density of the scanning station should be further optimized to achieve a higher measurement accuracy and efficiency.In the next step, the bamboo pole segmentation algorithm in the point cloud processing process will be added, which can remove the bamboo pole point cloud through the morphological difference between the bamboo pole and the banana pseudo-stem, and improve the measurement accuracy of banana plant count and pseudo-stem height. In addition, other banana phenotypic parameters, such as leaf length, leaf width, and leaf area, can be measured using 3D point cloud data. In future research, it will be extended to the measurement of other phenotypic parameters to improve the utilization efficiency of point cloud data.(1) Banana plants during the nutritional growth period were used as the research object. TLS was used to perform field-level banana plant number counting, banana single plant segmentation, and rapid measurement of pseudo-stem diameter and height.(2) By counting the K value in K-means clustering, the single plant segmentation of the canopy closed banana plant point cloud was realized, and the number of banana plants were counted automatically.(3) The cylinder segmentation method was used to measure the diameter of the banana pseudo-stem.(4) The sliding window along the axis of the banana pseudo-stem was used to identify the junction position between the banana pseudo-stem and the leaf. The junction position between the banana pseudo-stem and the ground was obtained, and the banana pseudo-stem height was measured.


Calorie restriction (CR) and exercise training (EX) are two critical lifestyle interventions for the prevention and treatment of metabolic diseases, such as obesity and diabetes. Brown adipose tissue (BAT) and skeletal muscle are two important organs for the generation of heat. Here, we undertook detailed transcriptional profiling of these two thermogenic tissues from mice treated subjected to CR and/or EX. We found transcriptional reprogramming of BAT and skeletal muscle as a result of CR but little from EX. Consistent with this, CR induced alterations in the expression of genes encoding adipokines and myokines in BAT and skeletal muscle, respectively. Deconvolution analysis showed differences in the subpopulations of myogenic cells, mesothelial cells and endogenic cells in BAT and in the subpopulations of satellite cells, immune cells and endothelial cells in skeletal muscle as a result of CR or EX. NicheNet analysis, exploring potential inter-organ communication, indicated that BAT and skeletal muscle could mutually regulate their fatty acid metabolism and thermogenesis through ligands and receptors. These data comprise an extensive resource for the study of thermogenic tissue molecular responses to CR and/or EX in a healthy state. Both larities . Consistlarities . Skeletalarities ,5. BAT elarities ,7. NeverCR, a classical example of negative energy balance, extends healthy lifespan from yeast to mammals ,9,10. ThExercise training (EX) causes many adaptations in the body that contribute to beneficial effects on health, including enhancing insulin sensitivity and reducing circulating lipid concentrations, primarily via adaptations to skeletal muscle ,17,18. REnhanced skeletal muscle or BAT activities play essential roles in the treatment of obesity. Skeletal muscle can produce cytokines, including proteins, peptides, enzymes and metabolites, and these cytokines can contribute to the weight loss induced by EX ,30. In aThus, in this study, we carried out RNA-seq of BAT and skeletal muscle to investigate the molecular and pathway alterations in response to CR and/or exercise interventions. To explore whether CR and EX have synergistic benefits, we restricted the EX mice to 70% calorie intake.Four-week-old healthy male C57BL/6J mice were purchased from GemPharmatech Co., Ltd. All mice were housed under standard laboratory conditions (12 h on/off) and in a temperature-controlled environment (22–24 °C) in the SPF Animal Research Center of Shanghai University of Sports (SYXK 2014-0002). The mice were given ad libitum access to food and water or fed a CR diet beginning at 8 weeks of age. The glucose fed to mice was tested three days before they were sacrificed. Mice were sacrificed 24 h after the last bout of EX. Mice in all groups were fasted for 6 h prior to tissue harvesting. Tissues were then rapidly dissected and processed or stored for analysis. Compared to the baseline food intake, the food intake of the mice was reduced by 10% per week to a final level of 70% of the ad libitum food intake at the 8th week. Eight-week-old male C57BL/6J wild-type mice with and without CRs were randomly divided into the sedentary and EX groups. The treadmill treatment included 2 days of adaptive training. For the adaptive EX, the treadmill was set at a 10° incline and began with a 5 min 0 m/min acclimation period, followed by 10 m/min for 10 min and 14 m/min for another 10 min. Mice started to run on the third day . Mice were fasted for 3 h prior to EX. A MiniSpec MQ10 nuclear magnetic resonance analyzer (Bruker) was used to measure the body composition of the mice according to the manufacturer’s instructions. Briefly, mice were put in a nuclear magnetic resonance tube and loaded in the machine. The body composition was measured automatically by the machine.&E images.BAT tissues were fixed in a 4% paraformaldehyde solution for at least 24 h and embedded in paraffin. Hematoxylin and eosin (H&E)  staining was undertaken. The tissues were processed as per routine for paraffin embedding, and 5 μm thick sections were cut and placed on glass slides. The paraffin-embedded sections were dewaxed with xylene, washed with a gradient of ethanol to water and then incubated with hematoxylin and eosin  for 4 min and sealed after conventional ethanol dehydration. Finally, the sections were analyzed under a Nikon light microscope at the indicated magnification. ImageJ software was used to calculate the areas of droplets in HA total of 15 BAT samples, including 3 from the sedentary group (control), 4 from the CR group, 4 from the EX group (EX) and 4 from the CR + EX group (CREX), and 14 gastrocnemius muscle tissue samples, including 4 from the control group, 3 from the CR group, 4 from the EX group and 3 from the CREX group, were used to perform transcriptome analysis. The Trizol method was used to extract total RNA according to the manufacturer’s instructions. RNA quality was measured using an Agilent 2100 Bioanalyzer . A previously reported method was utilized to construct cDNA libraries for each sample . Librarip value < 0.05 and |log2FC| > log2(1.5). Then, GO and KEGG pathway analyses of the DEGs were carried out using the enrichGO and enrichKEGG functions in the R package clusterProfiler (v4.6.0).The raw sequencing data were filtered with trim-galore (v0.6.7) by removing reads containing sequencing adapters and reads with low-quality bases. The clean reads were mapped to the reference genome (mm10) using STAR (v2.7.10a). Quantification of gene expression was calculated using rsem (v2.0.1) with the following command: . We conducted a differently expressed (DE) genes analysis for the two groups. Subsequently, differential expression analysis for the groups was carried out using DESeq2 (v1.38.0) with an adjusted Based on previous secretome profiling data from adipose tissue and skeletal muscle obtained with the LC-MS/MS method, we determined the DEGs encoding adipokines and myokines. Then, we performed GO and KEGG analyses to explore the roles of these DEGs using the enrichGO and enrichKEGG functions in the R package clusterProfiler (v4.6.0) We input gene counts from the bulk RNA-seq data from BAT and GAS, as well as the multi-subject single-cell profiles from previously published single-cell RNA sequencing (scRNA-seq) or single-nuclei RNA sequencing (snRNA-seq) data , into the MuSiC2 algorithm in R4.2.2. Cell types from the scRNA-seq and snRNA-seq were based on prior categorizations. Genes in the bulk RNA-seq data and single-cell and single-nuclei profiles used gene symbols as their identifiers. MuSiC2 used cell-type-specific gene expression from scRNA-seq data to characterize cell type compositions in the bulk RNA-seq. The estimated proportions were normalized to sum to 1 across the included cell types. The Wilcoxon test was used to calculate comparisons between group levels. https://zenodo.org/record/3260758 (accessed on 8 January 2023). Ligands regulated by CR with or without EX for which at least one specific receptor was expressed ) in the receiver tissue  were used for the next analyses. Then, NicheNet (v1.1.1) was used to rank the ligands based on how well they predicted whether a gene was linked with a gene set of interest compared to the background gene set. The gene sets of interest for the BAT and skeletal muscle were defined as metabolic-related DEGs. All other genes expressed in the receiver tissue  were considered background. Ligand activity scores were calculated as the Pearson correlation coefficient for the ligand–target regulatory potential scores of each selected ligand and the target indicator vectors, which indicated whether a gene belonged to the gene set of interest or not. For the top five ligands with the highest ligand activity, the corresponding receptors were exhibited in a ligand–receptor heatmap. Furthermore, the most prominent target genes for the top five ligands were chosen according to the regulatory potential score, with the genes presented relating to the indicated gene set of interested and being among the 5000 most strongly predicted targets of at least one of the top five ligands. Ligand–target gene interactions were illustrated in a circle plot using the R-package circlize (v0.4.15). To analysis ligand activity in the two tissues, we first defined the set of potentially active ligands in the ‘‘sender’’ tissue . Ligand–receptor interactions and weighted-networks data were downloaded from Unpaired one-tailed t tests or Wilcoxon rank sum and signed rank tests were performed to compare the two groups. One-way ANOVA and post hoc Tukey’s multiple comparison tests were performed for intergroup comparisons of more than two groups.We studied 8-week-old C57BL/6J male mice treated with CR and/or treadmill running exercise training (EX) interventions for 8 weeks B. The reTo investigate the molecular alterations, we conducted transcriptomic analysis of BAT under the CR and/or EX conditions. The principal component analysis plots of the different interventions are shown in Next, we evaluated the similarities and differences between CR and CREX with regard to transcriptomic alterations. There were 222 upregulated and 341 downregulated genes overlapping in the CR and CREX comparisons. While 522 downregulated and 277 upregulated genes were only found in the CR comparison, 130 downregulated and 88 upregulated genes were found in the CREX comparison J. AnalysTranscriptome analysis demonstrated that EX caused a few genes to change, which was consistent with the lack of changes in BW. PCA plots of the EX vs. control groups and CREX vs. CR groups are displayed in BAT can secrete various adipokines executing autocrine, paracrine and endocrine regulatory functions . We explTo profile transcriptional alterations in skeletal muscle response to CR and/or EX, bulk RAN-seq of skeletal muscle was performed. The principal component analysis plot for the four-group intervention is shown in Next, we assessed the similarities and differences in transcriptomic alterations caused by CR and CREX I. There Then, we analyzed the role of CR-specific regulated DEGs. Upregulated DEGs participated in the lipid metabolic process and the biosynthesis of amino acids, and downregulated DEGs were primarily involved in ECM–receptor interactions, insulin resistance and lipid metabolism . CREX-spSimilarly to the BAT transcriptome, a few DEGs were identified in skeletal muscle upon EX with or without CR. The principal component analysis plots of the EX versus control groups and CREX versus CR displayed no significant differences . DEG anaSkeletal muscle has been reported to secret myokines to communicate with other tissues ,39,40,41GO and pathway analyses showed that co-upregulated myokines were mainly enriched in functions associated with calcium ion transport and the ECM, while co-downregulated DE myokines were primarily related to cell growth, cell migration, cellular response to environmental stimuli and cancer-related pathways D,E. CR-sBAT and skeletal muscle are heterogeneous tissues with different cell types. We analyzed the cell proportions in BAT and skeletal muscle. To recognize the cell population compositions of the two tissues, we performed deconvolution analysis using published single-cell or single-nuclei RNA sequencing data . Statistical comparisons were not performed for proportions of cell subgroups close to or equal to zero. For the BAT, five cell populations—adipose cells, myogenic cells, mesothelial cells, endothelial cells and smooth muscle cells—were analyzed A. Only mBAT and skeletal muscle originate from the same precursors in the somites and display multifaceted interactions. Our group previously reported that BAT could communicate with skeletal muscle via myostatin . Others Next, we investigated whether CR-regulated, skeletal muscle-derived ligands had the potential to regulate BAT-selective gene programs related to fatty acid metabolism and thermogenesis F. The toFurthermore, we performed NicheNet analyses of the CREX and EX conditions. The top five BAT-derived ligands with the highest predicted activity in the CR with EX condition were Apoe, Bmp5, Col5a3, Pdgfd and Sfrp2, and all had several receptors with high interaction potential in skeletal muscle . These rSkeletal muscle and BAT are two well-described thermogenic sites that utilize distinct mechanisms for heat production. CR and EX are two lifestyle interventions that aim to produce improvements in metabolic health. The comparative effects of EX vs. CR on transcriptomics in BAT vs. skeletal muscle have not previously been reported. In the present study, we compared the effects of 8 weeks of CR with/without EX on mRNA expression and signaling pathways relating to thermogenesis, metabolism and ECM; in addition, we examined the cell populations and potential ligand–receptor communications between BAT and muscle. Our data showed that CR and CREX can significantly reduce body weight and fat mass; however, we did not observe significant differences between the EX and control groups. CR, EX and CREX could decrease fed glucose; however, there was a greater decrease in the CREX group compared to the EX group. Our findings highlight that strategies targeting obesity should combine both CR and EX. White adipose tissue, accounting for the largest volume of adipose tissue in humans, is critical for energy storage, endocrine communication and glucose homeostasis . BrowninEX and CR can reduce weight loss by increasing skeletal muscle and BAT burning; however, evidence has shown that exercise alone without dietary restrictions cannot reduce body weight significantly, which indicates that exercise alone is not effective for weight loss, especially for those patients with cardiometabolic diseases . Our stuUnexpectedly, the present study did not detect significance in the ability of EX to alter mRNA levels of markers of lipid or carbohydrate metabolism in BAT or skeletal muscle. Several reasons may account for this. First, the time of tissue collection: mice were sacrificed 24 h after the last bout of running when the mRNA profiles were probably back to normal. Second, the intensity of exercise: we trained the mice with a low-intensity recipe, which might not have been sufficient to cause gene expression alterations. Third, the duration of running: the mice only ran 1 h per day. Longer duration and higher intensity for the exercise are required in further investigations.Our results in


Retinal ischemia, often associated with various disorders such as diabetic retinopathy (DR), retinal vein occlusion, glaucoma, optic neuropathies, stroke, and other retinopathies, is a major cause of visual impairment and blindness worldwide. As proper blood supply to the retina is critical to maintain its high metabolic demand, any impediment to blood flow can lead to a decrease in oxygen supply, resulting in retinal ischemia. In the pathogenesis of DR, including diabetic macular edema (DME), elevated blood glucose leads to blood-retina barrier (BRB) disruptions, vascular leakage, and capillary occlusion and dropouts, causing insufficient delivery of oxygen to the retina, and ultimately resulting in visual impairment. Other potential causes of DR include neuronal dysfunction in the absence of vascular defect, genetic, and environmental factors. The exact disease progression remains unclear and varies from patient to patient. Vascular leakage leading to edema clearly links to visual impairment and remains an important target for therapy. Despite recent advances in the treatment of DME and DR with anti-VEGFs, effective therapies with new mechanisms of action to address current treatment limitations regarding vessel regeneration and reperfusion of ischemic retinal areas are still needed. The Wnt signaling pathway plays a critical role in proper vascular development and maintenance in the retina, and thus provides a novel therapeutic approach for the treatment of diabetic and other retinopathies. In this review, we summarize the potential of this pathway to address treatment gaps with current therapies, its promise as a novel and potentially disease modifying therapy for patients with DR and opportunities in other retinal vascular diseases. Diabetic retinopathy (DR) remains a leading cause of vison loss in many developed countries and is the most common, specific, and severe microvascular ocular complication of diabetes. According to The International Diabetes Federation, 463 million people were diagnosed with diabetes in 2019 ,6. In thVascular endothelial growth factor (VEGF) is a prominent factor inducing pathological neovascularization and altering retinal capillary permeability ,12,13. AHowever, as these therapies focus on the vascular leakage and inflammation component of the disease, their impact on vessel regeneration and reperfusion of ischemic retinal areas are minimal. Since vessel regeneration and a reduction of the retinal ischemic area could potentially lead to a durable reduction in hypoxia-induced signaling factors such as VEGF, effective therapies with this mechanism of action would be highly desirable. The Wnt signaling pathway is essential for embryonic development and in adults for tissue homeostasis and injury repair. This pathway has been shown to be critical for proper vascular development and for the maintenance of BRB in the adult retina ,29. DysrThe Wnt signal transduction cascade is a key driver of numerous biological events throughout the life of all animals . The act1–10), and 2 LRPs (LRP5 and LRP6) [The canonical Wnt signaling pathway, referred here as Wnt/β-catenin pathway is the most widely studied Wnt signaling pathway and it involves WNT binding to a heterodimeric receptor complex comprised of a frizzled (FZD) family of 7-pass transmembrane receptors and a single pass transmembrane co-receptor, low-density lipo-protein receptor-related protein (LRP). There are 19 mammalian WNTs, 10 FZDs (FZDnd LRP6) ,39. FollNDP (Norrie Disease Protein) gene, is secreted by Müller glia and endothelial cells, and plays a critical role in the patterning of the retinal vasculature during development and the establishment of the BRB during adulthood [4 and LRP5 in the presence of the Norrin-specific co-receptor tetraspanin 12 (TSPAN12), forming a ternary complex that activates the Wnt/β-catenin signal in retinal endothelial cells [NDP mutations result in Norrie disease, an X-linked genetic disorder characterized by hypovascularization of the retina, retinal detachment, and severe visual impairments or loss of vision [4FZD, LRP5, or TSPAN12 mutations are also the causes of a spectrum of related congenital retinopathies such as osteoporosis-pseudoglioma syndrome, familial exudative vitreoretinopathy (FEVR), and Coats disease, each of which share resembling phenotypes with Norrie disease. Mutations in 4Fzd, Lrp5, Tspan12, or Ndp in mice result in retinopathies with remarkable similarity to human diseases, including aberrant development of the retinal vasculature, BRB defects, and impaired vision [Significant evidence has demonstrated essential roles for Wnt/β-catenin signaling in both retinal and central nervous system (CNS) angiogenesis during development and in establishing and maintaining BRB/blood-brain-barrier (BBB) function in adults. In the retina, Norrin, encoded by the dulthood ,41,42,43dulthood ,45, is aal cells ,47  in humans, which may cause cataracts, glaucoma, intraocular hemorrhages, retinal detachment, and visual impairment ,60. Wnt74 in adult mice results in the loss of barrier function in the retina as well as in the cerebellum and olfactory bulb. Acute induction of Norrin in adult Ndp null mice restores cerebellar barrier function. Moreover, constitutively active β-catenin in endothelial cells in the choroid plexus causes upregulation of claudin-5 (Cldn5), a marker normally expressed in an intact BBB, and downregulation of Plvap, a marker of fenestrated BBB [Wnt/β-catenin signaling also plays a critical role in the maintenance of barrier function in both the retinal and the CNS vasculature in adults. Acute loss of Fzdated BBB ,62. Furtated BBB .Given the essential roles of Wnt/β-catenin signaling in the development of the retinal vessels and the maintenance of adult vessel barrier functions, it is not surprising that dysregulated Wnt/β-catenin signaling has been observed in ocular diseases. For example, total β-catenin levels were reported to be elevated in non-proliferative DR patients  while th4/LRP5/TSPAN12 signaling pathway plays an indispensable role in retinal vascular development and BRB integrity suggests that modulating this pathway may have therapeutic potential to treat retinal vascular diseases. Indeed, overexpression of lens-derived Norrin via gene delivery not only completely restored normal vascular development in Norrin-deficient (y/−Ndp) mice [The fact that Norrin/FZD/−) mice , but it /−) mice . The pot/−) mice . These r4 signaling activation, whereas the expression of Plvap is downregulated [4 signaling inhibits vascular leakage. In addition, the intravitreal injection of Norrin in streptozotocin-induced diabetic rats partially, but significantly reduced retinal leakage [4-specific Wnt/β-catenin pathway is a potential target as a new therapeutic strategy for the treatment of retinal vascular diseases including DR.In addition to inducing proper vessel regeneration, Norrin also reduced retinal vascular permeability ,69,73. Eegulated ,64,68,734/LRP5/TSPAN12 signaling pathway has a significant potential to treat retinal vascular disease, successful pharmacologic FZD4 activation through recombinant Norrin for therapeutic BRB modulation and retinal vessel regeneration has remained elusive. Norrin is poorly secreted and is highly associated with the extracellular matrix [4, LRP5/6, and TSPAN12 [Although Norrin/FZDr matrix , making  TSPAN12 ,76. SincActivation of Wnt/β-catenin requires the binding of WNT ligands to FZD receptors and to the co-receptors LRP5/6. WNTs bind to the cysteine-rich domain (CRD) on the N-terminus of FZD via two binding sites that have been described as “thumb” (site 1 on FZD) and “index finger” (site 2 on FZD), and the critical palmitoleoyl group on WNT is involved in site 1 binding . LRP5/6 4 on site 2 and LRP5/6 on the E1E2 domain [4 mutation, M157V [Tspan12 KO mice show stunted retinal vessel development [Tspan12 KO mice (US10035854). Treatment with P6C.51.61 in a mouse OIR model also showed trends of reduced pathological neovascular tufts formation and partially induced vascular regrowth in the vaso-obliterated avascular area. These results support the approach of activating Norrin/FZD4 signaling for the treatment of retinopathy. However, one of the limitations of this enhancer approach is the reliance on the presence of endogenous ligands. In addition, other Wnt ligands such as Wnt3a, Wnt7a, and Wnt10a are present in the vitreous humor, and some have been reported to be upregulated in OIR [Norrin engages FZD2 domain ,76. Simin, M157V  and a Non, M157V , both idn, M157V , and Tspelopment . P6C.51.d in OIR . Therefo4/LRP5 mediated Wnt/β-catenin signaling in endothelial cells. Unfortunately, WNT ligands are even more difficult to work with than Norrin due to their lipid modification that makes them insoluble and difficult to express, and their lack of FZD selectivity. However, recent breakthroughs have allowed for the development of FZD-specific WNT-mimetic molecules based on various protein scaffolds [Another way to enhance Wnt signaling in the retina is through the direct pharmacological activation of FZDcaffolds ,83,84,854 with another diabody containing an LRP5 E1 and an LRP5 E3 binder in the tetravalent trispecific configuration [4-mediated Wnt/β-catenin signaling to a similar extent as recombinant Norrin in HEK293 cells. Unlike Norrin, F4L5.13-induced signaling is not TSPAN12-dependent. Treatment of a mouse brain endothelial cell line (bEnd.3 cells) in vitro by F4L5.13 led to rescue of tight junction proteins Zo-1, Cldn3, and Cldn5 expression on the cell surface that was reduced by the pretreatment of these cells with VEGF and led to reduced permeability of the cells in vitro [Tspan12 KO mice with F4L5.13 restored retinal angiogenesis and barrier function as indicated by increased Cldn5 and decreased Plvap expression. Similar to what was observed with anti-LRP5 Norrin enhancer antibody described above, F4L5.13 treatment also reduced pathological neovascular tufts formation in the mouse OIR model [One approach to design WNT mimetics is based on a tetravalent diabody format where two diabodies, each having two binding sites for FZD or LRP, are tethered on two ends of an Fc fragment . Becauseguration . F4L5.13in vitro . Since FIR model . HoweverIR model .Another approach to WNT mimetic generation has focused on other antibody-based formats. Using scFv based fragments, different design parameters, including valency, linker lengths, relative orientation, geometry, stoichiometry, and receptor specificity, were first systematically evaluated . This le4/LRP5 specific WNT mimetic molecule, SZN-413 (4 with high affinity (with KD < pM) and the VHH domains bind LRP5. SZN-413 is highly potent, capable of activating Wnt/β-catenin signaling in vascular endothelial cells such as HRMEC and bEnd.3 cells in vitro and its potency is several orders of magnitude higher than that of recombinant Norrin or WNT3A in vitro. SZN-413 has biophysical and chemical characteristics that translate to desirable solubility, stability, and large scale manufacturability. Similar to F4L5.13, the bispecificity toward FZD4/LRP5 may have the advantage of averting promiscuous activation of other FZD family members in the vitreous space as both FZD4 and LRP5 are enriched in retinal endothelial cells vs. other cell types [4/LRP5 specific WNT mimetic molecule may be able to transform pathologic neovascularization into physiological neovascularization, reducing retinal ischemia and the ensuing overproduction of signaling molecules responsible for the development of pathologic neovascularization in the proliferative stage of diabetic retinopathy. This regeneration of vessels could be disease modifying, and has the potential to have a durable treatment effect; however, this will need to be confirmed in the clinic. In addition, dose–response data have shown that efficacy could be obtained at concentrations as low as 40 nM with a maximum effect at or above 4 µM suggesting that high doses may further extend the durability provided safety is not compromised. More research on the efficacy duration of SZN-413 is needed to determine the dose and delivery frequency of the drug. Even though both SZN-413 and F4L5.13 should be based on a similar mechanism of action the avascular area size reduction was not observed with F4L5.13 in OIR [Recently, a FZD SZN-413 C, was coll types . SZN-413ll types . The uprll types ,73, by Sll types . Further3 in OIR . The rea4/LRP5 specific WNT mimetic, inhibits the VEGF-driven retinal leakage in rabbit eyes [165, intravitreally injected into rabbit eye); considering that the VEGF concentration seen in proliferative DR or DME patients is less than 10 ng/mL in vitreous humor [Although inhibition of VEGF-driven retinal leakage is a major consideration for retinopathy treatment, studies to date have not sufficiently provided direct evidence showing WNT ligands can successfully perform this function in vivo. Recently, Diaz-Coranguez et al. showed that intravitreal injection of recombinant Norrin can inhibit the VEGF-driven retinal leakage in rats . Consistbit eyes . Moreoveus humor ,89,90, SGSK-3β is a key intracellular component of the Wnt/β-catenin signaling pathway. In the absence of WNT ligands, a destruction complex consisting of AXIN/GSK-3β/CK1/APC phosphorylates β-catenin leading to its ubiquitination and degradation. Upon ligand binding to FZD/LRP, the destruction complex containing GSK-3β is recruited to the intracellular side of the receptor complex on the membrane via DVL and AXIN, leading to stabilization of β-catenin and subsequent activation of Wnt target genes . Therefo4/LRP5/TSPAN12 axis has been shown to be essential for the proper formation of retinal vascular structure during development, and for the maintenance of proper vascular structure and function in adults. Activation of this pathway using several different approaches, including the use of Norrin, Norrin enhancing anti-LRP5 antibodies, FZD4 activating WNT mimetics, and GSK-3β inhibitors all suggest that activation of Wnt/β-catenin signaling could be a novel strategy for the treatment of retinal vascular diseases such as DR. Common findings from these Wnt activators are an increase in tight junction protein expression, a reduction in vascular leakage in vitro and/or in vivo, and a reduction in pathological neovascular tufts formation in the ischemic mouse OIR model. These findings are consistent with the established role of Wnt/β-catenin signaling in tight junction protein expression and the maintenance of the BRB in adult vessels [Wnt/β-catenin signaling mediated through the Norrin/FZD vessels . In addi vessels , suggestTspan12 KO mice, it did not induce vessel regrowth in the ischemic mouse OIR model [4) shuts down the vicious cycle of vascular break down, hypoxia, production of factors inducing pathological angiogenesis, and restores normal tissue healing process and regenerates physiological vasculatures. It would be exciting to explore whether these healing effects are indeed observed in the clinical setting. This novel mechanism of action of FZD4/LRP5 specific WNT mimetic opens new possibilities to treat diabetic and potentially other retinopathies. Although choroidal vessels do not have tight junctions, the observation that activation of Wnt/β-catenin signaling in choroidal endothelial cells reduces permeability [4 activating WNT mimetics may also provide a benefit for wAMD.The effects on vessel regeneration are somewhat variable between these Wnt activating approaches. While F4L5.13 rescued the vascular insufficiency in IR model  whereas IR model  and the IR model . It is nIR model ,97,98. Feability  offers a4 activating WNT mimetics could also have benefits in treating inherited human vitreopathies. Many mutations identified in these patients are located in TSPAN12, 4FZD, or LRP5 that affect Norrin binding or Norrin induced receptor complex formation [4 M157V and Norrin C95R FEVR mutants by P6C.51.61 and since FZD4 activating WNT mimetics do not rely on TSPAN12 to signal and could bind to different regions on FZD4 and LRP5 from Norrin, FZD4 activating WNT mimetics could effectively induce Wnt/β-catenin signaling in individuals harboring these mutations and offer a potential option for treating these inherited retinal vascular diseases.FZDormation ; therefo4/LRP5 in retinal vascular endothelial biology, and expression analysis suggest that FZD4/LRP5 may provide more selectivity toward the retinal endothelial cells over other cell types in the vitreous space, other FZDs have been reported to be expressed on retinal endothelial cells [4. Given the critical role of Wnt/β-catenin on brain endothelial function and maintenance of the BBB, FZD4 activating WNT mimetics could find broader applications for conditions characterized by a disrupted BBB such as stroke, epilepsy, and certain neurodegenerative diseases. In conclusion, modulation of Wnt/β-catenin signaling, especially mediated through FZD4 activating WNT mimetics, may offer a novel therapeutic approach for retinal vascular diseases such as DR and other various vascular diseases.While human genetics highlight the importance of FZDal cells . Future


Caenorhabditis elegans cadherin and catenin shows that these proteins are key for embryonic morphogenesis, we know surprisingly little about their structure and attachment to the cytoskeleton. In contrast to mammalian α-catenin that functions as a dimer or monomer, the α-catenin ortholog from C. elegans, HMP1 for humpback, is a monomer. Our cryogenic electron microscopy (cryoEM) structure of HMP1/α-catenin reveals that the amino- and carboxy-terminal domains of HMP1/α-catenin are disordered and not in contact with the remaining HMP1/α-catenin middle domain. Since the carboxy-terminal HMP1/α-catenin domain is the F-actin-binding domain (FABD), this interdomain constellation suggests that HMP1/α-catenin is constitutively active, which we confirm biochemically. Our perhaps most surprising finding, given the high sequence similarity between the mammalian and nematode proteins, is our cryoEM structure of HMP1/α-catenin bound to F-actin. Unlike the structure of mammalian α-catenin bound to F-actin, binding to F-actin seems to allosterically convert a loop region of the HMP1/α-catenin FABD to extend an HMP1/α-catenin FABD α-helix. We use cryoEM and bundling assays to show for the first time how the FABD of HMP1/α-catenin bundles actin in the absence of force. Collectively, our data advance our understanding of α-catenin regulation of cell–cell contacts and additionally aid our understanding of the evolution of multicellularity in metazoans.The regulation of cell–cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to filamentous actin at cell–cell junctions. The cadherin–catenin complex plays key roles in cell physiology, organism development, and disease. While mutagenesis of  Cell–cell contacts are essential in the maintenance of the architecture of tissues and the communication between cells. At the center of these cell–cell junctions is the cadherin receptor. Upon binding to calcium, the extracellular domain of one cadherin binds to that of a neighboring cell. In the cytosol, the transmembrane cadherin binds β-catenin, which binds the cytoskeletal protein α-catenin. The resulting cadherin-catenin complex is key to cell physiology, organism development, and disease , 6, 7, 8Caenorhabditis elegans, proteins equivalent in structure and function to mammalian cadherin, β-catenin, and α-catenin are HMR1, HMP2, and HMP1, respectively  . The amiDespite over 39% sequence identity and 60% similarity along the lengths of human α-catenin and HMP1/α-catenin polypeptide chains, HMP1/α-catenin is a monomer , while mKey known α-catenin structures are the complex between α-catenin and β-catenin , dimericC. elegans has an α-catenin homolog, named HMP1 for humpback. HMP1/α-catenin shares over 39% sequence identity throughout the human α-catenin polypeptide chain . While vertebrate α-catenins are monomeric or dimeric depending on their location and function, the roundworm HMP1/α-catenin does not dimerize  α-catenin as a control that, in comparison, shows traces of possible dimerization as analyzed by size exclusion chromatography (SEC) and multiangle light scattering .The nematode dimerize  . We used the zebrafish (D. reiro) and the slime mold (Dictyostelium discoideum) α-catenin as controls.Loss of functions mutants of HMP1/α-catenin cause circumferential F-actin bundles to detach from the plasma membrane. This process results in the folding of the dorsal epidermis and the Humpback phenotype . HMP1/α-Fig. 1g) analyses allowed F-actin bundling readouts compared with high-speed sedimentation  analyses. In the absence of actin binding proteins, F-actin remains in the supernatant under low-speed conditions while F-actin pellets upon high-speed centrifugation.Truncations of the FABD of α-catenin affect its binding to F-actin . To put E and 2A). Recent total internal reflection fluorescence microscopy showed that bundling by the mammalian α-catenin FABD was increased upon motor activation (B and C). However, we only observed F-actin bundling when we assayed with the FABD but not with full-length HMP1/α-catenin (E). Further, full-length monomeric human α-catenin did also not bundle F-actin while full-length dimeric human α-catenin bundled F-actin (F). However, when truncating the carboxy terminus on the human α-catenin FABD (Δ886–906 or Δ866–906) bundling is no longer observed compared to the human α-catenin FABD that was not truncated (residues 660–906) (G).We observed that the HMP1/α-catenin FABD bundled F-actin E and 2A.tivation . Interestivation , B and C-catenin E. Furthe F-actin F. Howeve660–906) G.Figure D), we only observed partial decoration of F-actin as determined by 2D classification. The partial decoration consistently led to 3D reconstructions that lacked density for HMP1/α-catenin. Extensive efforts to enrich the decoration through careful curation of 2D classes did also not lead to decorated HMP1/α-catenin actin filaments.Although full-length HMP1/α-catenin binds F-actin D, we onlA).On the other hand, the isolated HMP1/α-catenin FABD bundles F-actin  which haFig. 4B). For example, Val-809 of one HMP1/α-catenin interacts with Ile-872 of a separate HMP1/α-catenin polypeptide chain.As seen for the binding of mammalian α-catenin, the carboxy terminus of HMP1/α-catenin extends to engage in hydrophobic interactions with HMP1/α-catenin of a neighboring HMP1/α-catenin B. For exC). As seen with our human α-catenin crystal structure . Specifically, Trp-711 predicted to be at the end of the first α-helix in the unbound HMP1/α-catenin FABD structure (C), that is also seen in our unbound human α-catenin structure (D). When bound to F-actin, Trp-711 fills the pocket that Trp-861 occupies in the unbound model. HMP1/α-catenin residue Trp-861 corresponds to human α-catenin residue Trp-859 (B). In the F-actin bound human α-catenin structure (E), Trp-859 fills the pocket similarly as for the equivalent HMP1/α-catenin Trp-861 (C). The extension of the second α-helix, by borrowing residues from the loop region, no longer leaves room for the loop region of the last α-helix that therefore becomes disordered in the actin-bound HMP1/α-catenin structure . This disorder is consistent with SEC-small-angle X-ray scattering (SAXS) data that showed that the FABD connects via a flexible linker (residues 630–675) to the middle domain  were not detected in 2D classification. This suggests that the middle domain of HMP1/α-catenin is the only part rigid enough to align during 2D classification, while the rest of HMP1/α-catenin is flexible A. This de domain .5h5m) (B). Similarly, the truncated murine αN-catenin structure  (C). While the truncated HMP1/α-catenin crystal structure has one polypeptide chain in the asymmetric unit (space group P 212121), the truncated αN-catenin structure has two polypeptide chains in the monoclinic (P 21) crystal. Thus, despite the different crystal contacts in the nematode versus murine middle domain structures, these can be superimposed with 1.8 Å root-mean-square deviation for 1,647 atoms. Given that the two crystal lattices resulted in similar interdomain interactions, it seems that in full-length HMP1/α-catenin, the interdomain interaction differs by ∼20 Å movements, as seen in our cryoEM structure (B and C).In comparison to our cryoEM HMP1/α-catenin structure, the truncated HMP1/α-catenin crystal structure   has two 5h5m) B. Similary 5xfl)  also hasry 5xfl) C. While tructure , B and C6upv) or murine (PDB entry 6wvt) α-catenin to F-actin (6upv) or murine (PDB entry 6wvt) α-catenin, respectively  that mediates longitudinal actin-actin interactions , 37. Ade F-actin , 18. Speectively .Molecular insights into α-catenin are crucial to understanding α-catenin regulation of cell–cell contacts. While a wealth of mammalian α-catenin structural data is available, structural insights into HMP1/α-catenin are limited. Thus, our HMP1/α-catenin studies provide significant new structural data and will additionally aid our understanding of the evolution of multicellularity in metazoans.Despite the high sequence identity amongst α-catenins from different species, only some of these proteins dimerize. Our full-length HMP1/α-catenin cryoEM structure shows that HMP1/α-catenin is a highly flexible monomeric protein. Several phosphorylation sites have been identified in HMP1/α-catenin . In contIn contrast, the phosphomimetic and nonphosphorylatable mutant of Ser-649 localized to cell–cell junctions as seen for wild type HMP1/α-catenin middle domain . This isThe lack of interdomain interactions in our full-length HMP1/α-catenin is relevant to its functions. First, the amino-terminal HMP1/α-catenin domain that binds HMP2/β-catenin at cell–cell junctions is disordered in our structure and therefore displays considerable conformational freedom. This allows HMP1/α-catenin to readily bind to partners like HMP2/β-catenin without prior activation. Similarly, we find that the carboxy-terminal FABD of HMP1/α-catenin is not interacting with the other HMP1/α-catenin domains. In contrast to mammalian α-catenin, HMP1/α-catenin is therefore constitutively active in linking HMR1/cadherin to the actin cytoskeleton. Indeed, there are also differences in the affinity of the various α-catenins with filamentous actin , 41.SEC-SAXS data showed that monomeric human αE-catenin is a compact globular structure with a maximum dimension of about 147 Å  that is Human α-catenin was shown to bind F-actin cooperatively , 44, 45.We found that full-length HMP1/α-catenin co-sedimented with filamentous actin. A S823F mutant has previously been shown to reduce the binding of HMP1/α-catenin with F-actin . This isFurthermore, the isolated HMP1/α-catenin FABD resulted in heavy F-actin bundling, as seen by our cryoEM analyses. Such bundling was not observed by full-length HMP1/α-catenin. Our findings are consistent with an earlier report that showed that HMP1/α-catenin co-sedimented with F-actin significantly less compared to the isolated HMP1/α-catenin FABD . Our cryThe carboxy terminus is disordered in our F-actin-bound FABD HMP1/α-catenin cryoEM structure and in our dimeric full-length human crystal structure. Given that carboxy-terminal truncations prevent bundling, it seems that the last ∼20 residues are responsible for bundling. The AlphaFold  model ofIt is tempting to speculate that in the full-length monomeric structure, this carboxy-terminal region might be buried in intermolecular interactions and thus prevent bundling.Although our actin-bound sample for cryoEM structure determination comprises HMP1/α-catenin residues 677 to 927, the HMP1/α-catenin amino terminus (residues 677–710) was disordered in our cryoEM map. This is consistent with the finding that HMP1/α-catenin Δ677-703 localized to cell–cell junctions as seen for wild type HMP1/α-catenin . HoweverC. elegans are just beginning to be uncovered  were cloned into the pET28 vector to have an amino-terminal octa-Histidine tag followed by a PreScission protease site immediately before the inserts using the following primers:pET28 vector:Forward: TGAGAATTCGCGGCCGCACTCGAGCACCACReverse: CATATGGGGCCCCTGGAACAGAACTTCCAGATGHuman α-catenin FABD (upper case is used for the vector and lower case for the insert):660 Forward: CTGGAAGTTCTGTTCCAGGGGCCCcatgacgatcagctgatagctggcc906 Reverse: CGGCCGCGAATTCTCAgatgctgtccatagctttgaactcgctg885 Reverse: CGGCCGCGAATTCTCAtgcccgtttaatcttggtctgtgtctcatc865 Reverse: CGGCCGCGAATTCTCActctggtgccttcatcttccatgD. discoideum α-catenin was procured as an amino-terminally tagged GST fusion with a tobacco etch virus cleavage site (Dicty stock center).The HMP1/α-catenin FABD (residues 677–927) and full-length human α-catenin (residues 1–906) were cloned into pGEX-6P-1 with an amino-terminal GST tag and a PreScission protease cleavage site by DNA Custom Cloning. The expression construct of g for 15 min and stored at −80 °C.Constructs were transformed into BL21(DE3) (Novagen) and expressed in 2 L of Luria-Bertani media at 37 °C until the absorbance at 600 nm reached 0.6 to 0.8 and induced with 1 mM isopropyl β-D-1-thiogalactopyranosides for 20 h at 25 °C. Cells were pelleted by centrifugation at 5000Escherichia coli BL21(DE3) pellets containing amino-terminally GST-tagged HMP1/α-catenin were thawed and resuspended in lysis buffer containing 400 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, and lysed by sonication (75% amplitude with 3 min processing time and 5 s on followed by 10 s off). The lysate was clarified by centrifugation  and then loaded onto a GST prep 16/10 FF chromatography column that was equilibrated with 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, and 2 mM β-mercaptoethanol. Elution was completed with 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, 2 mM β-mercaptoethanol, and 10 mM reduced glutathione. Peak elution fractions were pooled and dialyzed overnight in 2 L of buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 0.5 mM EDTA, and 2 mM β-mercaptoethanol, after the addition of 200 μg PreScission protease. The sample was then reloaded onto a GST prep 16/10 FF column. The flowthrough containing cleaved HMP1/α-catenin was collected, concentrated to 2 mg/ml, and aliquoted into 1 ml aliquots after adding glycerol , and stored at −80 °C.To generate purified full-length HMP1/α-catenin, frozen Prior to cryoEM grid preparation, an aliquot was thawed and passed through a Superdex S200 10/300 Gl column equilibrated in 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 7.5), and 1 mM dithiothreitol. The purification of the FABD of HMP1/α-catenin was performed as described for full-length HMP1/α-catenin.D. rerio, and D. discoideum α-catenins were purified similar to HMP1/α-catenin using 1 mM dithiothreitol instead of β-mercaptoethanol. Cleavage of the GST tag for D. discoideum α-catenin was carried out with tobacco etch virus protease.Full-length human, For full-length human α-catenin, the monomer and dimer peaks were pooled separately, and a second SEC run was performed of each pool on a Superdex S200 10/300 Gl column pre-equilibrated in 20 mM tris(hydroxymethyl)aminomethane (pH 8), 150 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine.E. coli BL21(DE3) pellets containing amino-terminally octa-Histidine-tagged α-catenin FABDs were thawed, resuspended in 500 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 1 mM β-mercaptoethanol, and lysed by sonication for 3 min (5 s on and 10 s off cycles). The lysate was clarified by centrifugation at 100,000g for 45 min and filtered. The supernatant was loaded onto two HisTrap HP 5 ml columns in tandem equilibrated in a buffer containing 150 mM NaCl and 20 mM tris(hydroxymethyl)aminomethane (pH 8) on an AKTA FPLC at a flow rate of 1 ml/min. The column was then washed thoroughly with 30 column volumes of buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 50 mM imidazole. Bound protein was then eluted using a gradient to 500 mM imidazole. The eluted protein fractions were pooled and subjected to overnight PreScissoin protease treatment (200 μg) with dialysis against a buffer containing 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), 1 mM ethylenediaminetetraacetic acid, and 1 mM dithiothreitol. The PreScissoin protease treated α-catenin FABD proteins were concentrated and loaded onto a Superdex 75 26/60 column equilibrated in 150 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (pH 8), and 1 mM dithiothreitol. The peak fractions were concentrated and stored at −80 °C.For purification of the human α-catenin FABD proteins , frozen We used SEC and multiangle light scattering analyses to determine the absolute masses with an Agilent 1260 Infinity HPLC with a variable wavelength detector for UV absorption monitoring which is coupled in line with Dawn-Heleos II multiangle light-scattering detector (Wyatt Technology) and OptiLab T-rex differential refractive index detector (Wyatt Technology). We loaded our samples on an analytical Superdex S200 10/300 Gl column that was pre-equilibrated with 20 mM tris(hydroxymethyl)aminomethane (pH 8), 150 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine, at a flow rate of 0.5 ml/min. We used bovine serum albumin (Pierce) as a standard and performed data acquisition and analyses using ASTRA software version 6.1.2, 0.5 mM β-mercaptoethanol) at 4 °C. We polymerized G-actin (at 0.6 mg/ml) by adding the polymerization buffer  to fully polymerize overnight. F-actin was stored at 4 °C for up to 2 weeks.We dialyzed rabbit skeletal muscle G-actin (50 μl at 1 mg/ml) into 500 ml of G-actin buffer (2 mM tris(hydroxymethyl)aminomethane [pH 8], 0.2 mM adenosine triphosphate, 0.2 mM CaCl2, 10 mM MgCl2, 100 mM KCl, and 10 mM adenosine triphosphate) for binding assays or in KMEI buffer , 2 mM adenosine triphosphate, and 0.5 mM ethylene glycol tetraacetic acid) and used for bundling assays. For binding assays, F-actin (15 μM) was incubated with 8 μM full-length proteins of HMP1/α-catenin, zebrafish (D. reiro) α-catenin, or slime mold (D. discoideum) α-catenin. For F-actin bundling assays, F-actin (4 μM) was incubated with 3 μM of full-length HMP1/α-catenin or 15 μM of HMP1/α-catenin FABD proteins. After incubation at room temperature for 15 min, the samples were spun at appropriate speeds  for 15 min. The supernatant and the pellet were analyzed on sodium dodecyl-sulfate polyacrylamide gel electrophoresis and the bands visualized by Coomassie R-250 staining. As controls, F-actin bundling with full-length α-catenin (residues 1–906) monomer and dimer along with α-catenin FABDs  were also analyzed.For F-actin co-sedimentation assays, rabbit muscle actin (Cytoskeleton Inc) was freshly polymerized either in Tris buffer (20 mM tris(hydroxymethyl)aminomethane [pH 8.0], 0.2 mM CaClFor the HMP1/α-catenin FABD in complex with F-actin cryoEM structure determination, C-Flat 1.2/1.3400 mesh grids were glow discharged for 60 s at 15 mA using a PELCO easiGlow (Ted Pella Inc) and mounted on Leica GP2 cryogenic plunger (65% humidity and 21 °C). First, 3.5 μl of F-actin (0.7 μM) was placed on the carbon film and incubated for 60 s and subsequently successive addition and removal of 3.5 μl of 0.6 mg/ml (21 μM) HMP1/α-catenin. The addition and removal steps were repeated twice before blotting from the backside of the grid for a total of 12 s. The grids were immediately plunge frozen in liquid ethane that is maintained at -183 K and cooled by liquid nitrogen. Lower concentrations of the HMP1/α-catenin FABD were unsuitable as they resulted in partial decoration. The grids were transferred into a Japan Electron Optics Laboratory (JEOL) cryoARM300 for screening and data collection.For the full-length HMP1/α-catenin cryoEM structure determination, an Au-Flat 1.2/1.3300 mesh grid (Protochips) was glow discharged at 15 mA for 240 s using a PELCO easiGlow (Ted Pella Inc) and taken into Leica GP2 cryogenic plunger set to 95% humidity and 4 °C. About 4 μl of 0.3 mg/ml of full-length HMP1/α-catenin was applied on the carbon film side, blotted on the carbon side for 7 s, and plunged immediately for vitrification in liquid ethane and transferred into a JEOL cryoARM300.All cryoEM data were recorded on a JEOL cryoARM300 operated at 300 kV. The illumination was set to a spot size of three and an angle of four at a magnification of 60,000. The condenser aperture was set to 50 μm with an Omega in-column energy filter, a slit width of 20 eV, and a zero-loss peak that aligned every 6 h. Electron micrographs were collected using serialEM  on a K3 Raw movies were imported into the cryoSPARC  interfacFor the FABD of HMP1/α-catenin bound to F-actin, 20,737 micrographs were collected to compensate for the high degrees of bundling observed in many micrographs and to improve the final resolution. After micrograph curation, 3576 images were selected, while the rest were removed. Since the 2D classes exhibited a high degree of decoration, a larger box size was not needed to separate unbound from bound particles. Instead, an extraction box size of 1080 pixels (399 Å) was used, and 2D classification was only used to separate suitable particles from unsuitable particles. After helical refinement, CTF refinement, and nonuniform refinement, a reconstruction of a 3D map at 3.36 Å resolution from 1,990,015 particles was achieved (EMDB entry EMD-26805), with a helical twist of −166.9°, and a helical rise of 27.72 Å.ab initio volume calculation. The final homogenous refinement yielded a 4.8 Å resolution reconstruction from 149,802 particles (EMDB entry EMD-26748).For full-length HMP1/α-catenin, a total of 2781 movies were collected, and after curation, 1294 micrographs were selected for further processing. Select 2D classes obtained from the initial round of blob picking and extraction were used to carry out the second round of template-based particle picking. The resultant 2D classes were used to select suitable particles for initial 7uuw).To determine the cryoEM structure of the HMP1/α-catenin FABD bound to F-actin, the cryoEM structure of our human α-catenin (residues 22–906) bound to F-actin was first fitted into the map using Chimera. A HMP1/α-catenin homology model was generated with SWISS-MODEL  and then5h5m) was initially docked into our cryoEM map. The structure was better fitted after chain refinement using COOT (To determine the cryoEM structure of full-length HMP1/α-catenin, the crystal structure of the middle domain of HMP1/α-catenin (PDB entry ing COOT .7uuw. The cryoEM reconstructions for the HMP1/α-catenin FABD bound to F-actin and the unbound HMP/α-catenin were deposited in EMDB with accession codes EMD-26805 and EMD-26748, respectively. All other data are within the manuscript.The coordinates for the HMP1/α-catenin FABD bound to F-actin are deposited with the Protein Data Bank, accession code This article contains The authors declare no conflict of interest with the contents of this article.