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All rights reserved The expected tariff cost is significantly lower than the $4 billion to $5 billion crosstown rival General Motors estimates which Ford attributes to its higher mix of U.S.-built vehicles Metrics details The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field Here we report the development of OptoShroom3 an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels Its application to murine and human neural organoids leads to thickening of neuroepithelia These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context this approach has been mainly applied to study cell-level events and its application to induce morphogenesis in complex tissue shapes is technically challenging Since all previous tools have been based on recruitment to the plasma membrane they require a precise multi-photon stimulation of the apical membrane to induce constriction of the apical side only there is still a lack of tools to manipulate 3D tissue deformation and reconstitute mammalian morphogenesis offers unique opportunities to study the interplay between tissue shape and function in vitro the manipulation of organoid shape with optogenetic tools remains unexplored is capable of fast activation and deactivation of apical constriction at the cell level We demonstrate that an increase in apical tension causes tissue folding and lumen shrinkage in epithelial cell sheets and neural organoids a Protein structure of wild-type Shroom3 and scheme of apical constriction b Design of OptoShroom3 constructs that dimerize upon blue light stimulation c MDCK cells expressing GFP-NShroom3-iLID and SspB-mCherry-CShroom3 before and after 1-min stimulation d Non-muscle myosin IIb stainings of OptoShroom3 MDCK cells under no stimulation (left) and 2-h blue light stimulation (right) e Single-cell stimulation cycles and representative images from constriction experiments Stimulated area was designed as the initial apical area of the cell Right panel shows a color-coded compounded image comparing the start and end of stimulation g Quantification of the apical area in stimulated (N = 17) and non-stimulated (N = 16) cells The area was normalized to the last measurement before stimulation (t = 5 min) h Quantification of apical and basal areas during 3 periods of 10 min of stimulation and rest (N = 8 The basal slice was defined as ~5 μm below the apical slice The areas were normalized to the last measurement before stimulation (t = 10 min) These results suggest that OptoShroom3 induces apical constriction by causing a redistribution of non-muscle myosin II in the apical junctions a Apical slice of iRFP-CAAX signal of MDCK cells on glass The white box marks the stimulated area (26.5 × 26.5 μm) b Reconstruction of a 3D segmented MDCK monolayer c Cell group stimulation cycles and representative images from constriction experiments Lateral slice of iRFPCAAX and 3D segmentation d Color-coded compounded image comparing the start and end of stimulation and volume measurements for 2 h stimulation of OptoShroom3 MDCK cells separating between stimulated cells and the rest of the cells in the frame (outer area) (NExperiments = 5 j Model prediction of cell height based on average measurements shown in panel g k Re-analysis of apical area reduction classifying cells by layers (NExperiments = 5 predictions accurately matched the measured dynamics while being offset from the real values The offset could be due to an underestimation in volume measurements an increase in cell height can be expected when volume and basal area are conserved This could be due to light diffusion weakly activating the layers surrounding the illumination area As no layer of cells displayed expansion of the apical area we hypothesized that OptoShroom3 may provoke cell displacements to accommodate the gradient of apical constriction a Apical slice of iRFP-CAAX signal of MDCK cells on a thick collagen gel b Averaged PIV analysis on apical slices before and after stimulation in a 35.3 × 35.3 μm area (N = 6) c Division of PIV frame in three areas for analysis d Temporal evolution of radial vector components averaged by area Positive vectors represent movements towards the center of stimulation (N = 6 e Integration of vectors averaged by area (N = 6 f Apical slice of iRFP-CAAX signal of MDCK cells on a collagen gel The white rectangles mark two stimulated areas g Averaged PIV analysis on apical slice during (2 min) and after stimulation of two 20 × 170 μm areas (N = 7) a Protocol for the formation of flat MDCK monolayers in a thick matrigel bed using acetic acid incubation (top) and scheme of measured parameters (bottom) b 3D renders and higher resolution lateral slices of representative colony folding before and after 24 h of stimulation (stimulated cycle: 3 min off 57 min on; non-stimulated cycle: 3 min on (GFP-NShroom3-iLID image acquisition) c Skeletons used for curvature measurements of representative examples of non-stimulated (left) and stimulated C450V mutant (right) colonies d Quantification of changes in average curvature of stimulated stimulated C450V mutant and non-stimulated colonies Concave (apical) curvature was set as positive (NStimulated  = 7 e Z-projected images of colonies used for area measurements of non-stimulated (left) f Quantification of relative changes in the projected area of stimulated and non-stimulated colonies (NStimulated = 7 g Representative example of irreversible colony folding on matrigel labeled with infra-red fluorescent beads White arrowheads indicate matrigel breaking points and yellow arrowhead indicates matrigel being deformed and carried over with the folding colony h Quantification of relative changes in the projected area of stimulated colonies during and after stimulation (stimulation period in blue) (N = 5 These results show that OptoShroom3 can irreversibly provoke tissue curvatures and folds in a spatiotemporal manner and that tissue folding is achieved through deformation and remodeling of matrigel a The formation and stages of mouse optic vesicle organoids b Representative example of stimulation of neuroepithelium (day 5 Arrow indicates the translocation of SspB-mCherry-CShroom3 c Thickness measurements of neuroepithelia before and after stimulation (days 4–8) (N = 19 d Representative example of optic vesicle organoid on day 8 e Stimulation cycles and representative example of 488 nm stimulation of an optic vesicle showing transmitted light and GFP-NShroom3-iLID signal f Measurements of lumen diameter of optic vesicles before and after OptoShroom3 stimulation (days 6–8) (N = 27 g Quantification of the lumen diameter change for control (non-stimulated) and stimulated samples after 55 min (NNon-stimulated = 17 Boxplot boundaries first to third quartile h The formation stages of human neuroectodermal organoids i GFP-NShroom3-iLID expression on a neuroectodermal organoid on day 6 j Representative example of OptoShroom3-induced flattening through local stimulation of neuroectodermal organoids The center of the stimulated area was set up as 0 degrees l Curvature measurements of neuroectodermal organoids before and after stimulation (N = 8 These results demonstrate that OptoShroom3 can manipulate morphologies of organoids The outcome of deformation depends on initial tissue geometry and the forces to which the tissue is already subjected to we have developed an optogenetic tool to control apical constriction in mammalian tissues The tool induced a quick reduction of the apical cell surface (starting 1 min after stimulation) and the stimulation of groups of cells provoked cell elongation and collective displacements in the apical surface of adjacent cells Apical constriction in epithelial colonies on soft gels induced the irreversible folding of cell sheets apical constriction thickened the neuroepithelium and reduced the inner lumen when the inner surface was apical it flattened the tissue when the outer surface of organoids was apical These results illustrate how the induction of apical constriction in different contexts can lead to different changes in tissue structure The presented experiments support the idea that OptoShroom3 induces actomyosin constriction specifically on the apical side and that cell elongation is a consequence of the induced apical constriction reported that stimulation longer than 5 min caused an irreversible reduction of cell junctions OptoShroom3-induced apical constriction and cell elongation in MDCK cells on glass were reversible independently of the duration of the stimulation OptoShroom3-induced colony folding was irreversible and provoked changes in the matrigel substrate These results suggest that when OptoShroom3-induced force can be transduced to a deformable substrate the recruitment of OptoShroom3 is specific to the apical junctions of epithelial cells because of the specific and continuous localization of GFP-NShroom3-iLID OptoShroom3 does not require subcellular precision of stimulation by multi-photon microscopy to induce apical constriction Even if a whole cell is stimulated by a scanning laser or an LED SspB-mCherry-CShroom3 will be recruited to the apical side and induce constriction in that area only This is advantageous especially to stimulate tissue with complex shapes such as the apical surface of an optic vesicle OptoShroom3 cannot induce actomyosin constriction in basal or lateral areas or in cells that do not possess apicobasal polarity novel tools that can induce morphological changes will be necessary not only to study in vivo morphogenesis but also to reproduce the morphogenetic processes in vitro We expect that OptoShroom3 will provide a new avenue towards understanding how tissue shape and mechanotransduction affect cell fate and differentiation given their inherent complexity and accessibility stand out as perfect candidates for the application of these new morphogenetic tools to further study the interplay between tissue shape and function OptoShroom3 constructs GFP-NShroom3-iLID and SspB-mCherry-CShroom3 have been submitted to Addgene (catalog numbers: 170976 and 170977 iRFP-CAAX and GFP-CAAX sequences were prepared through traditional cloning methods iRFP gene was acquired from Addgene (piRFP CAG promoter was used for OptoShroom3 and GFP-CAAX MDCK cells were maintained in Dulbecco’s Minimal Essential Medium (DMEM) with high glucose Cells were split every 2–3 days through 9-min incubation with EDTA 50 mM pH = 7.4 and 3 min with 0.25% trypsin (Gibco) Mouse ES cell line (EB5, Rx-GFP) was described previously41 mES cells were maintained on gelatin-coated dishes with DMEM supplemented with 15% fetal bovine serum The 2i medium was used to increase the number of optic vesicles per organoid Cells were split every 2–3 days with 0.25% trypsin Human iPS cell line ((IMR90)−4) was purchased from WiCell hiPS cells were maintained in StemFlex media (Gibco) on matrigel-coated dishes DMEM-F12 (Gibco) with 1.23% matrigel (Corning #356231) was used for coating for 30 min at 37 °C Cells were split every 4–5 days with Accutase (STEMCELL Technologies) and StemFlex media was supplemented with ROCK inhibitor Y-27632 (10 µM) during the first day after splitting Since using publicly available human iPS cells constitutes acceptable practice in EMBL the ethics evaluation of the project was officially exempted by the institute PiggyBac plasmids were introduced using Amaxa Nucleofector (Lonza) stable clones were picked for every cell line All cell lines were cultured at 37 °C in a humidified incubator with 5% CO2 and regularly tested for mycoplasma contamination MDCK samples for immuno-fluorescence staining were prepared by seeding 2 × 105 MDCK cells on a 12 mm diameter glass (113 cells/mm2) Whole sample stimulation was carried out 2 days after seeding with an array of blue LEDs inside a cell-culture incubator Samples were placed on top of the LED array with a white methacrylate diffuser to make light homogeneous cells were fixed at 37 °C and 5% CO2 maintaining the stimulation or darkness condition Primary and secondary antibody incubations were carried out for 1 h at room temperature Antibodies and probes used were: rabbit Non-muscle Myosin Heavy Chain II-B Antibody (dilution 1:200 alexa fluor 647 goat anti-rabbit (dilution 1:200 and alexa fluor 647 goat anti-mouse (dilution 1:200 Apical and basal area measurements in Fig. 1 were conducted on a custom-made python pipeline applying watershed segmentation to iRFP-CAAX confocal stacks of MDCK cells on glass-bottom dishes Basal stacks were selected as ~5 µm below apical Projected area and skeletons for curvature measurements of folding MDCK colonies were obtained from confocal stacks using a custom-made python segmentation pipeline For the analysis of non-muscle myosin IIb intensity in immunofluorescence stainings the GFP-NShroom3-iLID signal was used to segment the apical junctions the average intensity per pixel of non-muscle myosin IIb in the junctional area was divided by the average intensity per pixel in the cytoplasmic area (considered as the remaining pixels in the image) the measurements showed minimal differences with the advantage of our custom pipeline being fully automated Cell volume was calculated through voxel counting of the segmented cells Cell height was measured by finding the distances between the average height of contact with the apical and the basal side Apical and basal areas were measured through the acquisition of a 2D image of the cell voxels in contact with either the apical or basal side Cells were tracked from frame to frame based on maximum overlap quality control of the segmentation was carried out by discarding cells that were not present in all frames of the timelapse Cells that had sudden changes in volume higher than 30% (mis-segmented dividing and apoptotic cells) were also eliminated from the analysis For the classification shown in Fig. 2e–h and Supplementary Fig. 5 all cells in contact with the stimulation area were considered as stimulated only cells with more than 50% of their apical area inside the illumination area at the start of the stimulation were considered stimulated 2 × 105 MDCK cells were seeded on a 12 mm-diameter glass (113 cells/mm2) Samples were stimulated 2 days after seeding as described in live imaging methods Translocation measurements were carried out with FIJI using apical slices of MDCK cells A 0.362 μm2 area (5 × 5 pixels) of the junctional or cytoplasmic region was manually selected and averaged for every time point Three areas for each type (junction or cytoplasm) were selected on every image each junction measurement was divided by the closest cytoplasm measurement a stable GFP-CAAX and doxycycline-inducible mCherry clonal cell lines were mixed at 5% ratio with a GFP-CAAX stable cell line Samples were induced with 0.5 μg/ml 24 h before the measurement only the GFP-CAAX stable cell line was used Local curvature was defined as the inverse of the radius of the osculating circle to three points of a curve Curvature was measured by calculating the osculating circle of three equally distanced points (20 pixels distance) in the skeleton equivalent to calculating the inverse of the radius of a circumscribed circle around a triangle: Being R the radius of the osculating circle of points A and CA are the distance between the points Local curvatures were measured along the skeleton of the main axis and averaged for every time point to obtain a general curvature measurement SspB-mCherry-CShroom3 slices were segmented Curvature measurements were applied to the perimeter of the segmented organoids distance between points was set up higher (320 pixels) to filter out curvatures caused by very small features The cross-correlation method was used with 1 pass with an interrogation window size of 64 pixels and experiment averaging were performed with a custom python pipeline 20 μl of 100% matrigel (Corning #356234) were set on glass-bottom dishes and solidified at 37 °C for 10 min gels were incubated with 20 mM acetic acid (90 μl 4.5 matrigel-acetic acid volume ratio) at 37 °C for 40 min gels were washed twice with PBS and 4 × 105 MDCK cells were seeded in 80 μl of DMEM medium More media (2 ml) was added 1.5 h after seeding These parameters were empirically obtained to make isolated flat MDCK colonies which were stimulated 2 days after seeding Stimulated colonies were illuminated for 57 min every hour The remaining 3 min were used for the acquisition of GFP-NShroom3-iLID signal in control colonies (non-stimulated colonies) We considered the impact of this period of illumination on control colonies and of lack of illumination on stimulated colonies negligible Colonies that showed migratory behavior were not considered for the analysis a final concentration of 0.05% infra-red fluorescent beads (Thermo Fisher Scientific dark red 660/68) was added to matrigel prior to polymerization All matrigel samples for stiffness measurements were prepared following the same procedure as described for tissue folding settings Then the gels were covered with PBS and measured using Chiaro Piuma nano-indenter with a probe of stiffness 0.027 N/m Measurements were analyzed using DataViewerV2 (Optics11 Life) Optic vesicle organoids were prepared following SFEBq method41 mES cells expressing OptoShroom3 were dissociated with 0.25% trypsin and quickly reaggregated in a differentiation medium G-MEM supplemented with 1.5% knockout serum replacement 3000 cells were cultured in 100 µl media per well of a 96 well low attachment U bottom plate (Thermo) Although the ES cells express the retinal marker Rx-GFP during the optic vesicle formation the Rx-GFP expression is much lower than that of GFP-NShroom3-iLID and thus negligible Measurements for neuroepithelial thickening and lumen reduction were manually performed using FIJI Each measurement was performed three times and averaged For Supplementary movie 11 2 µM SiR-tubulin (Spirochrome) was added to day-6 organoids and incubated for 1 h before stimulation The modifications from the commercial protocol were: 2000 cells were used as the starting number Y-27632 (10 µM) was added from day 0 to day 3 and the use of Accutase for cell disaggregation No statistical method was used to predetermine the sample size. All experiments were performed at least three times except for Supplementary movie 11 (two times) some MDCK colonies on matrigel presented a migratory behavior Because this migration made them move out of the stimulation and imaging area The Investigators were not blinded to allocation during experiments and outcome assessment Further information on research design is available in the Nature Research Reporting Summary linked to this article Source data are provided with this paper. Due to the large file size, raw image data can be obtained upon request. Please contact Miki Ebisuya or Guillermo Martínez-Ara to request data. A response will be provided in less than 2 weeks. Source data are provided with this paper Synthetic morphology: prospects for engineered Synthetic developmental biology: build and control multicellular systems Synthetic development: learning to program multicellular self-organization Synthetic Development: building mammalian multicellular structures with artificial genetic programs Apical constriction: a cell shape change that can drive morphogenesis Apical constriction: themes and variations on a cellular mechanism driving morphogenesis Synthetic biological approaches for optogenetics and tools for transcriptional light-control in bacteria Ruijgrok, P. V. et al. Optical control of fast and processive engineered myosins in vitro and in living cells. Nat. Chem. Biol. 1–9, https://doi.org/10.1038/s41589-021-00740-7 (2021) Illuminating developmental biology with cellular optogenetics Reverse and forward engineering multicellular structures with optogenetics Using optogenetics to tackle systems-level questions of multicellular morphogenesis Optogenetic manipulation of cellular communication using engineered myosin motors Illuminating cell signalling with optogenetic tools Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis Optogenetic control of cellular forces and mechanotransduction RhoA mediates epithelial cell shape changes via mechanosensitive endocytosis Single-Component Optogenetic Tools for Inducible RhoA GTPase Signaling Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis In vitro organogenesis in three dimensions: self-organising stem cells Organogenesis in a dish: modeling development and disease using organoid technologies Modeling development and disease with organoids a PDZ domain–containing actin-binding protein is required for neural tube morphogenesis in mice Shroom induces apical constriction and is required for hingepoint formation during neural tube closure Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut Developmental origins for kidney disease due to Shroom3 deficiency Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling Differential actin-dependent localization modulates the evolutionarily conserved activity of Shroom family proteins A new standard nomenclature for proteins related to Apx and Shroom Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins Factors that control the chemistry of the LOV domain photocycle Downregulation of basal myosin-II is required for cell shape changes and tissue invagination Araya García, C. A. in Reference Module in Biomedical Sciences (Elsevier, 2017). https://doi.org/10.1016/B978-0-12-801238-3.11055-4 Morphogenetic movements in the neural plate and neural tube: mouse Vertebrate intestinal endoderm development Self-organizing optic-cup morphogenesis in three-dimensional culture Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis Shroom family proteins regulate γ-tubulin distribution and microtubule architecture during epithelial cell shape change Cerebral organoids model human brain development and microcephaly Volume conservation principle involved in cell lengthening and nucleus movement during tissue morphogenesis Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation Engineering and application of LOV2-based photoswitches A toolbox to explore the mechanics of living embryonic tissues an important regulator of development and disease Mechanobiology of collective cell behaviours Cellular mechanotransduction: from tension to function Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells MorphoGraphX: A platform for quantifying morphogenesis in 4D Spatial organization of the extracellular matrix regulates cell–cell junction positioning Guided self-organization and cortical plate formation in human brain organoids Download references This work was supported by internal grants from RIKEN and EMBL; Grant-in-Aid for Scientific research (KAKENHI) programs from Japanese Ministry of Education and Technology (MEXT) (16H06170 and 18H04769 to M.E.); Research foundation for Opto-Science and Technology (to M.E.) is funded by European Research Council (Adv-883739) and La Caixa Foundation (LCF/PR/HR20/52400004) IBEC is a recipient of a Severo Ochoa Award of Excellence from the MINECO Matsuda for his help in cell-line preparation and discussions about OptoShroom3; to Y Sasai for his help in optic cup organoids; to A Marin for their helpful conversations on gel preparation and tissue folding; to L Batlle and the Tissue Engineering unit from the Center for Genomic Regulation for their instruction and comments in organoid culture; to M Benito-Kwiecinski for their instruction in the formation of neuroectodermal organoids and helpful suggestions; to M Gomez for his help in gel measurements and comments; to D Oriola for his help in the measurements of matrigel stiffness; to N Garreta for their support in experiment design and comments on the manuscript; to V Open Access funding enabled and organized by Projekt DEAL These authors contributed equally: Guillermo Martínez-Ara European Molecular Biology Laboratory (EMBL) Barcelona RIKEN Center for Biosystems Dynamics Research (RIKEN BDR) Institute for Bioengineering of Catalonia (IBEC) The Barcelona Institute for Science and Technology (BIST) Centro de Investigación Biomédica en Red en Bioingeniería Institució Catalana de Recerca i Estudis Avançats (ICREA) screened gene candidates for the induction of apical constriction and selected Shroom3 as a suitable candidate and tested optogenetic constructs as well as explored experimental settings for 3D tissue deformation helped the individual stimulation of MDCK OptoShroom3 cells helped with plasmid construction and cell line preparation wrote the manuscript with feedback from all authors The authors declare no competing interests Nature Communications thanks Jonathan Fouchard reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-022-33115-0 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Sign up for our daily Newsletter and stay up to date with all the latest news You are receiving this pop-up because this is the first time you are visiting our site You are using software which is blocking our advertisements (adblocker) we are relying on revenues from our banners So please disable your adblocker and reload the page to continue using this site.Thanks The Tudela artichoke season is currently in full swing as September and October have been much cooler and rainier than normal; nevertheless this cooler weather has allowed the plants to vegetate really well and we have kept potentially harmful pests under control without major problems so we are harvesting artichokes of very good quality," says Guillermo Agorreta highly appreciated both in haute cuisine and by the industry for its flavor and consistency remains one of Navarre's most important agricultural products despite the fact that its acreage has experienced a certain decline in recent years there has been a reduction in the number of hectares under production for various reasons One of them has been the emergence of the seed artichoke which is competing with the Blanca de Tudela variety but we see that people are once again opting for the classic Tudela artichoke because of its unique texture," says Agorreta the only artichoke suitable for the canning industry The processing industry and the high-end hospitality and catering sector are the most important market niches for the Tudela artichoke "The drop in the production has also happened as a result of the escalation in costs over the last few years which has made it necessary to adjust the sales prices and in Spain there is currently also a shortage of it so the combination of higher costs and the fact that it is very difficult to find qualified workers has led growers to reduce their acreage to facilitate a better management," says the head of the PGI "The truth is that the agricultural sector needs qualified and specialized personnel not only for cultivation but also to operate machinery and manage new technologies and it is difficult to find people with the right skills and the need for them will only increase," says Guillermo Agorreta I would say that we have reached a turning point we went through the most difficult period due to the escalation of costs but today we can say that the situation has stabilized quite a lot The industry has been able to assume the price increases that we needed to make and the demand from canning factories has increased the hotel and catering sector in Spain is doing spectacularly well and artichokes are a flagship food for many restaurants so the demand has also increased," says Agorreta "There already seems to be an upturn in the acreage and we hope that this trend will continue and that producers will be encouraged to expand their Tudela artichoke crops as consumers and top chefs undoubtedly prefer this product." For more information:Consejo Regulador de la IGP Alcachofa de Tudela (Tudela Artichoke PGI Regulatory Council)Avda Serapio Huici, 22. Edificio Peritos Villava/Atarrabia31610 - Navarre. SpainTel.: +34 948 01 30 45[email protected] FreshPublishers © 2005-2025 FreshPlaza.com (Via GoSanAngelo.com) A man and woman were killed in a crash early Tuesday afternoon on U.S 87 North about 4 miles north of Water Valley according to the Texas Department of Public Safety which was struck from behind by a 2015 Dodge Ram occupied by two men: driver Cesar Villava Garcia DPS spokesman Justin Baker said the motorcycle was traveling slowly when it pulled out of a rest area onto U.S The driver of the pickup saw the vehicle in the inside lane but then the slow-traveling motorcycle changed lanes resulting in the Dodge hitting it from behind were pronounced dead at the scene by Coke County Judge Roy Blair the Sterling County and Coke County Sheriff’s Offices and the Texas Department of Transportation responded to the crash 87 North near the Tom Green County-Coke County line according to the Texas Department of Public Safety.The man and woman were riding a 2006 Honda Shadow which was struck from behind by a Dodge Ram carrying two men.DPS spokesman Justin Baker said the motorcycle was traveling slowly when it pulled out of a rest area onto U.S resulting in the Dodge hitting it from behind.The man and woman on the motorcycle were pronounced dead at the scene The men in the pickup were not injured.No identities were being released because next of kin had not yet been notified.In addition to DPS the Sterling County and Coke County Sheriff’s Offices responded to the crash.No further information has been released Two people are confirmed dead in the crash: a man and woman who were on a motorcycle The Texas Department of Public Safety is investigating a crash on U.S The Tom Green County Sheriff's Office sent out an alert about 12:30 p.m Southbound lanes were still blocked as of1:20 p.m. with traffic delayed and rerouted to northbound lanes Officials had not yet released information by 1:30 p.m. but the crash appeared to have involved a motorcycle and an air conditioning work truck The footbridge of the river Arga, in the Pamplona Riverside Park, at the height of the Swimming Club is named after Maidemoiselle Agustini the artistic name of Remigia Echarren Aranguren a woman from Pamplona born in 1853 in Navarreria who at the end of the 19th century became the most famous tightrope walker of her time Although there is a street in Txantrea that bears her name in recognition of a pioneer woman the City Council has wanted to recognize Remigia Echarren in a place as emblematic as the Arga walkways next to the Caparroso Mill and with the artistic version of Remigia like Mademoiselle Agustini DISCOVER THE STORY OF REMIGIA ECHARREN The walkways were built in order to facilitate pedestrian traffic between the ever-growing district of La Magdalena and Villava/Burlada on which wooden boards were placed in order to cross the river When the river rose, however, it used to wash these boards away with it and so people crossed the river by jumping from block to block, as immortalised in Montxo Armendáriz’s film Secretos del corazón (Secrets of the Heart) though probably less charming footbridges in 2000 Although it is not known when she first performed in the city of Pamplona we do know that during the San Fermín festival of 1882 "La Echarren" as she was called was one of the main attractions in the bull ring with her act which consisted in crossing the ring from one side to the other on a tightrope fixed to the roof Pío Baroja was one of the amazed spectators who saw her that year Among the attractions offered during the San Fermín festival of 1883 particular mention should be made of her feat to cross the river Arga at a height of ten metres opposite the machine at the Pinaquy factory she did so with some small wicker baskets on her feet and retracing her path blindfolded and she received requests to perform year after year She also conquered the Plaza del Castillo square the City Hall gave her a donation of 500 pesetas to thank her for her performances even mentioned that: “On this memorable day Pamplona-born Remigia Echarren was proclaimed queen of the Arga or whatever you would like to call the river that waters the market gardens of the Rochapea Remigia Echarren is known in the world of the circus (…) by the stage name of Mlle The daringness of this funambulist was also reported in the local press The “Lau-buru” newspaper described the audacious feat as follows: Her skills made her famous beyond these borders The river Pisuerga in Valladolid and the estuary of Bilbao were other stages on which she demonstrated her circus skills She worked intensely between 1885 and 1892 the year in which she had an accident in Ondárroa when working on the tightrope with a chair at a height of fifteen metres She suffered a number of fractures which put an end to her working and professional life The lottery tickets she sold in the streets did not take her out of poverty increases the cost of a Discover Pass from $30 to $45 The Omak Elks Lodge hosted the grand coronation and fundraiser event for the 2025 Rodeo Champions of American (RCA) Washington Royalty The 3rd Annual Bonaparte Lake Ice Fishing Derby was a resounding success drawing 320 anglers and 439 class entries to the icy waters Miguel Indurain Larraya is a former Spanish road racing cyclist who has a net worth of $10 million He played a variety of sports growing up before discovering his passion for cycling He took second place in his first amateur race and became the youngest person to win the National Amateur Road Championship at 18 years old Indurain made his professional debut with Reynolds in 1984 after participating in the Los Angeles Olympics earlier in the year Indurain is just the fourth person in history to win five times in a row He's also a two-time winner of the Giro d'Italia who became famous for his impressive physiology His body took in oxygen at a rate nearly double that of the average person's Indurain announced his retirement in Pamplona and continues to be actively involved with cycling and the Olympics © 2025 Celebrity Net Worth / All Rights Reserved