Multi-chamber cardioids unravel human heart development and cardiac defects.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

The anterior chamber of the eye technology and its anatomical, optical, and immunological bases.

The anterior chamber of the eye (ACE) is distinct in its anatomy, optics, and immunology. This guarantees that the eye perceives visual information in the context of physiology even when encountering adverse incidents like inflammation. In addition, this endows the ACE with the special nursery bed iris enriched in vasculatures and nerves. The ACE constitutes a confined space enclosing an oxygen/nutrient-rich, immune-privileged, and less stressful milieu as well as an optically transparent medium. Therefore, aside from visual perception, the ACE unexpectedly serves as an excellent transplantation site for different body parts and a unique platform for noninvasive, longitudinal, and intravital microimaging of different grafts. On the basis of these merits, the ACE technology has evolved from the prototypical through the conventional to the advanced version. Studies using this technology as a versatile biomedical research platform have led to a diverse range of basic knowledge and in-depth understanding of a variety of cells, tissues, and organs as well as artificial biomaterials, pharmaceuticals, and abiotic substances. Remarkably, the technology turns in vivo dynamic imaging of the morphological characteristics, organotypic features, developmental fates, and specific functions of intracameral grafts into reality under physiological and pathological conditions. Here we review the anatomical, optical, and immunological bases as well as technical details of the ACE technology. Moreover, we discuss major achievements obtained and potential prospective avenues for this technology.

The MEK-ERK signaling pathway promotes maintenance of cardiac chamber identity.

Ventricular and atrial cardiac chambers have unique structural and contractile characteristics that underlie their distinct functions. The maintenance of chamber-specific features requires active reinforcement, even in differentiated cardiomyocytes. Previous studies in zebrafish have shown that sustained FGF signaling acts upstream of Nkx factors to maintain ventricular identity, but the rest of this maintenance pathway remains unclear. Here, we show that MEK1/2-ERK1/2 signaling acts downstream of FGF and upstream of Nkx factors to promote ventricular maintenance. Inhibition of MEK signaling, like inhibition of FGF signaling, results in ectopic atrial gene expression and reduced ventricular gene expression in ventricular cardiomyocytes. FGF and MEK signaling both influence ventricular maintenance over a similar timeframe, when phosphorylated ERK (pERK) is present in the myocardium. However, the role of FGF-MEK activity appears to be context-dependent: some ventricular regions are more sensitive than others to inhibition of FGF-MEK signaling. Additionally, in the atrium, although endogenous pERK does not induce ventricular traits, heightened MEK signaling can provoke ectopic ventricular gene expression. Together, our data reveal chamber-specific roles of MEK-ERK signaling in the maintenance of ventricular and atrial identities.

Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells.

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces - one composed of the atypical cadherin Fat2 (also known as Kugelei) and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptor Plexin A. Here, we show that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at leading edges of cells, but Lar and Semaphorin5c play little role in the localization of Fat2. Fat2 is also more stable at interfaces than Lar or Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. We propose that Fat2 serves as the organizer of this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.

Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments.

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Aberrant uterine folding in mice disrupts implantation chamber formation and alignment of embryo-uterine axes.

The uterine luminal epithelium folds characteristically in mammals, including humans, horses and rodents. Improper uterine folding in horses results in pregnancy failure, but the precise function of folds remains unknown. Here, we uncover dynamic changes in the 3D uterine folding pattern during early pregnancy with the entire lumen forming pre-implantation transverse folds along the mesometrial-antimesometrial axis. Using a time course, we show that transverse folds are formed before embryo spacing, whereas implantation chambers form as the embryo begins attachment. Thus, folds and chambers are two distinct structures. Transverse folds resolve to form a flat implantation region, after which an embryo arrives at its center to attach and form the post-implantation chamber. Our data also suggest that the implantation chamber facilitates embryo rotation and its alignment along the uterine mesometrial-antimesometrial axis. Using WNT5A- and RBPJ-deficient mice that display aberrant folds, we show that embryos trapped in longitudinal folds display misalignment of the embryo-uterine axes, abnormal chamber formation and defective post-implantation morphogenesis. These mouse models with disrupted uterine folding provide an opportunity to understand uterine structure-based mechanisms that are crucial for implantation and pregnancy success. This article has an associated 'The people behind the papers' interview.

Crystal structure and biochemical analysis suggest that YjoB ATPase is a putative substrate-specific molecular chaperone.

AAA+ ATPases are ubiquitous proteins associated with most cellular processes, including DNA unwinding and protein unfolding. Their functional and structural properties are typically determined by domains and motifs added to the conserved ATPases domain. Currently, the molecular function and structure of many ATPases remain elusive. Here, we report the crystal structure and biochemical analyses of YjoB, a Bacillus subtilis AAA+ protein. The crystal structure revealed that the YjoB hexamer forms a bucket hat-shaped structure with a porous chamber. Biochemical analyses showed that YjoB prevents the aggregation of vegetative catalase KatA and gluconeogenesis-specific glyceraldehyde-3 phosphate dehydrogenase GapB but not citrate synthase, a conventional substrate. Structural and biochemical analyses further showed that the internal chamber of YjoB is necessary for inhibition of substrate aggregation. Our results suggest that YjoB, conserved in the class Bacilli, is a potential molecular chaperone acting in the starvation/stationary phases of B. subtilis growth.

Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na+ restriction.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

Characterization of Fabry disease-associated lyso-Gb3 on mouse colonic ion transport and motility.

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by a deficiency in α-galactosidase A leading to the accumulation of globotriaosylceramide (Gb3) and subsequent increase in globotriaosylsphingosine (lyso-Gb3) in different cells and organs, including the gastrointestinal (GI) tract. GI symptoms represent some of the earliest manifestations of FD and significantly impact quality of life. The origin of these symptoms is complex, and the exact mechanisms remain poorly understood. Here, we sought to determine whether lyso-Gb3 contributes to the pathophysiology of GI symptoms associated with FD by examining its effects on mouse colonic ion transport and motility ex vivo using Ussing chambers and organ baths respectively. Lyso-Gb3 significantly increased colonic baseline short-circuit current (ISC). This increase in ISC was insensitive to inhibition of the cystic fibrosis transmembrane conductance regulator and Na-K-Cl cotransporter 1 suggesting that the increase in ISC is Cl- ion independent. This response was also insensitive to inhibition with the neurotoxin, tetrodotoxin. Additionally, pretreatment with lyso-Gb3 did not significantly influence subsequent responses to either veratridine or capsaicin implying that the response to lyso-Gb3 does not involve the enteric nervous system. In terms of colonic motility, lyso-Gb3 did not significantly influence colonic tone, spontaneous contractility or cholinergic-induced contractions. These data suggest that lyso-Gb3, significantly influences ion transport in mouse colon, but that accumulation of Gb3 may be a pre-requisite for the more pronounced disturbances in GI physiology characteristic of FD.

Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis.

The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein ß" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.

Chemosensory behaviour of juvenile crown-of-thorns sea star (Acanthaster sp.), attraction to algal and coral food and avoidance of adult conspecifics.

Intraspecific and habitat-mediated responses to chemical cues play key roles in structuring populations of marine species. We investigated the behaviour of herbivorous-stage juvenile crown-of-thorns sea stars (COTS; Acanthaster sp.) in flow-through choice chambers to determine if chemical cues from their habitat influence movement and their transition to become coral predators. Juveniles at the diet transition stage were exposed to cues from their nursery habitat (coral rubble-crustose coralline algae (CCA)), live coral and adult COTS to determine if waterborne cues influence movement. In response to CCA and coral as sole cues, juveniles moved towards the cue source and when these cues were presented in combination, they exhibited a preference for coral. Juveniles moved away from adult COTS cues. Exposure to food cues (coral, CCA) in the presence of adult cues resulted in variable responses. Our results suggest a feedback mechanism whereby juvenile behaviour is mediated by adult chemical cues. Cues from the adult population may deter juveniles from the switch to corallivory. As outbreaks wane, juveniles released from competition may serve as a proximate source of outbreaks, supporting the juveniles-in-waiting hypothesis. The accumulation of juveniles within the reef infrastructure is an underappreciated potential source of COTS outbreaks that devastate coral reefs.

Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Clinical validation of grass pollen exposure chamber in patients with allergic rhinitis triggered by timothy grass.

BACKGROUND: The fluctuation in concentrations of airborne allergens frequently presents a challenge to assessing the efficacy of allergen immunotherapy (AIT) in 'field' studies. Allergen exposure chambers (AECs) are specialized medical installations developed to expose individuals to allergens at defined and consistent concentrations under a controlled environment. The aim of the study was to validate the provocation test with timothy grass pollen as well as to assess its safety in the AEC in patients with allergic rhinitis. METHODS: In the ALLEC® AEC, varying concentrations of timothy grass pollen were dispersed. Allergic symptoms were measured by total nasal symptom score (TNSS), acoustic rhinometry, peak nasal inspiratory flow (PNIF) and nasal discharge volume. Lung function, assessed through peak expiratory flow rate (PEFR) and forced expiratory volume in the first second (FEV1), was used to evaluate safety. RESULTS: The consistency of the test was proved by the stability of environmental conditions, including temperature, humidity and CO2 levels, as well as constant concentrations of grass pollen at predetermined levels ranging from 1000 to 10,000 particles per cubic meter (p/m3). Allergic individuals developed symptoms at concentrations of 3000 p/m3 and above, across all measured endpoints. Lung function was not affected throughout all the challenges. The reproducibility of symptoms was confirmed throughout the tests. The concentration of 8000 p/m3 together with a challenge duration of 120 min was found to be optimal. CONCLUSION: The study demonstrates that the ALLEC® grass pollen exposure chamber provides a reliable and safe method for inducing repeatable symptoms in patients with allergic rhinitis. This approach can be effectively applied for allergy diagnostics and clinical endpoint determination during AIT.

Rhinoconjunctivitis severity induced by cat exposure influences early and late asthmatic responses: Evidence from an environmental exposure chamber.

BACKGROUND: The impact of allergic rhinoconjunctivitis on the early (EAR) and late asthmatic response (LAR) has yet to be assessed during optimal allergen exposure conditions. OBJECTIVE: We aimed to assess predictive factors of the EAR and LAR and to evaluate the relation between rhinitis, conjunctivitis and asthma induced by cat allergen exposure in an environmental exposure chamber (EEC). METHODS: Data from two cohort studies involving asthmatic patients with cat allergy who performed a cat allergen exposure challenge in ALYATEC EEC were analysed. Spirometry, visual analogue scale (VAS) for asthma, VAS for rhinitis, Total Nasal Symptoms Score, Total Ocular Symptoms Score (TOSS), Rhinoconjunctivitis Total Symptoms Score and Abelson score were used to assess asthma, rhinitis and conjunctivitis during and after exposure. RESULTS: An EAR occurred in 65.1% of patients, 32.1% of whom had a LAR. The diameter of the prick test to cat allergens and non-specific bronchial hypersensitivity level were independent risk factors for EAR (p < .05). No independent risk factors for LAR were identified. Rhinoconjunctivitis severity during exposure correlated with the asthma VAS during EAR and LAR (p < .05). Allergen exposure time needed to trigger an EAR correlated with the Abelson score during exposure (p < .05). The asthma VAS and TOSS during exposure correlated with faster LAR occurrence (p < .05). CONCLUSION: Prick test size and non-specific bronchial hypersensitivity level were confirmed as independent predictive factors of EAR during allergen exposure in an EEC. This study demonstrated the relation between the severity of rhinitis, conjunctivitis and asthma induced by allergen exposure for both EAR and LAR.

Anciently duplicated genes continuously recruited to heart expression in vertebrate evolution are associated with heart chamber increase.

Although gene/genome duplications in the early stage of vertebrates have been thought to provide major resources of raw genetic materials for evolutionary innovations, it is unclear whether they continuously contribute to the evolution of morphological complexity during the course of vertebrate evolution, such as the evolution from two heart chambers (fishes) to four heart chambers (mammals and birds). We addressed this issue by our heart RNA-Seq experiments combined with published data, using 13 vertebrates and one invertebrate (sea squirt, as an outgroup). Our evolutionary transcriptome analysis showed that number of ancient paralogous genes expressed in heart tends to increase with the increase of heart chamber number along the vertebrate phylogeny, in spite that most of them were duplicated at the time near to the origin of vertebrates or even more ancient. Moreover, those paralogs expressed in heart exert considerably different functions from heart-expressed singletons: the former are functionally enriched in cardiac muscle and muscle contraction-related categories, whereas the latter play more basic functions of energy generation like aerobic respiration. These findings together support the notion that recruiting anciently paralogous genes that are expressed in heart is associated with the increase of chamber number in vertebrate evolution.

Nonstructural carbohydrate dynamics' relationship to leaf development under varying environments.

Leaf-out in temperate forests is a critical transition point each spring and advancing with global change. The mechanism linking phenological variation to external cues is poorly understood. Nonstructural carbohydrate (NSC) availability may be key. Here, we use branch cuttings from northern red oak (Quercus rubra) and measure NSCs throughout bud development in branch tissue. Given genes and environment influence phenology, we placed branches in an arrayed factorial experiment (three temperatures × two photoperiods, eight genotypes) to examine their impact on variation in leaf-out timing and corresponding NSCs. Despite significant differences in leaf-out timing between treatments, NSC patterns were much more consistent, with all treatments and genotypes displaying similar NSC concentrations across phenophases. Notably, the moderate and hot temperature treatments reached the same NSC concentrations and phenophases at the same growing degree days (GDD), but 20 calendar days apart, while the cold treatment achieved only half the GDD of the other two. Our results suggest that NSCs are coordinated with leaf-out and could act as a molecular clock, signaling to cells the passage of time and triggering leaf development to begin. This link between NSCs and budburst is critical for improving predictions of phenological timing.

Enhanced decreases in rice evapotranspiration in response to elevated atmospheric carbon dioxide under warmer environments.

A short period of exposure to elevated CO2 is known to decrease evapotranspiration via stomatal closure. Based on theoretical evaluation of a canopy transpiration model, we hypothesized that this decrease in the evapotranspiration of rice under elevated CO2 was greater under higher temperature conditions due to an increased sensitivity of transpiration to changes in CO2 induced by the greater vapour pressure deficit. In a temperature gradient chamber-based experiment, a 200 ppm increase in CO2 concentration led to 0.4 mm (-7%) and 1.5 mm (-15%) decreases in 12 h evapotranspiration under ambient temperature and high temperature (+3.7°C) conditions, respectively. Model simulations revealed that the greater vapour pressure deficit under higher temperature conditions explained the variations in the reduction of evapotranspiration observed under elevated CO2 levels between the temperature treatments. Our study suggests the utility of a simple modelling framework for mechanistic understanding of evapotranspiration and crop energy balance system under changing environmental conditions.

Water Loss From Bagged Leaves During Storage: Why and When?

In ecophysiology leaves are frequently stored for hours after sampling before measuring their leaf water potential (Ψleaf). Here, we address a previously unidentified source of error, that metabolic heat generation can cause continuous water loss from leaves stored in impermeable bags, leading to a Ψleaf drop over time. We tested Ψleaf drop rates under various conditions: two bag materials, two species, initial Ψleaf above or below the turgor loss point (Ψtlp), and storage at 25°C versus 4°C. We partitioned leaf water loss due to condensation on the inner bag surface or permeation through the bag. We found that Ψleaf dropped by up to 0.39 MPa per hour, with 41%-89% of the water leaving the leaf condensed on the inner bag surface. Plastic bags showed higher Ψleaf drop rates than aluminium bags, and leaves above Ψtlp experienced greater drops. Storing leaves at 4°C reduced the Ψleaf drop rate by 60% compared to 25°C. Leaves were 0.2-0.3°C warmer than the bags, likely due to metabolic heating. Our energy balance model suggests that water loss is lower when storing leaves at cooler temperatures, using leaves with low stomatal conductance, deflated bags, and leaves with low Ψleaf.

The synergistic effect of multiple organic macromolecules on the formation of calcium oxalate raphides of Musa spp.

Needle-like calcium oxalate crystals called raphides are unique structures in the plant kingdom. Multiple biomacromolecules work together in the regulatory and transportation pathways to form raphides; however, the mechanism by which this occurs remains unknown. Using banana (Musa spp.), this study combined in vivo methods including confocal microscopy, transmission electron microscopy, and Q Exactive mass spectrometry to identify the main biomolecules, such as vesicles, together with the compositions of lipids and proteins in the crystal chamber, which is the membrane compartment that surrounds each raphide during its formation. Simulations of the vesicle transportation process and the synthesis of elongated calcium oxalate crystals in vitro were then conducted, and the results suggested that the vesicles carrying amorphous calcium oxalate and proteins embedded in raphides are transported along actin filaments. These vesicles subsequently fuse with the crystal chamber, utilizing the proteins embedded in the raphides as a template for the final formation of the structure. Our findings contribute to the fundamental understanding of the regulation of the diverse biomacromolecules that are crucial for raphide formation. Moreover, the implications of these findings extend to other fields such as materials science, and particularly the synthesis of functionalized materials.