Extracellular Matrix Material Research Articles

Hyaluronic Acid-Dopamine-NCSN Hydrogel Combined With Extracellular Matrix Promotes Wound Healing.

The skin barrier is essential to prevent pathogenic invasion. When injury occurs, multiple biological pathways are promptly activated and wound repair processes are triggered. The effective healing of wounds is essential for survival, and dysfunction could result from aberrant wound repair. Preparation of many hydrogels, which involve the addition of growth/cell factors or mimic extracellular matrix (ECM) components, has not resulted in significant advances in tissue recovery. ECM contains a large number of biologically active molecules that activate a variety of cellular transduction pathways, which are essential for wound repair. Here, this work prepares hyaluronic acid-dopamine-thiourea (HA-DA-NCSN) hydrogels exhibiting ultrafast gelation in situ, following the methods of Xu et al., and subsequently designs a hydrogel containing ECM particles. In addition, the loaded ECM material, specifically decellularized ECM material, not only enhances the strength of the hydrogel network, but also delivers bioactive substances that make it a suitable platform for skin wound repair. The ECM hydrogel has great potential as an efficient bioactive wound dressing. This research suggests that this strategy is likely to improve skin wound closure in rat skin wound models.

Constitutive Modeling of Mouse Arteries Suggests Changes in Directional Coupling and Extracellular Matrix Remodeling That Depend on Artery Type, Age, Sex, and Elastin Amounts.

Arterial stiffening occurs during natural aging, is associated with an increased risk of adverse cardiovascular events, and can follow different timelines in males and females. One mechanism of arterial stiffening includes remodeling of the extracellular matrix (ECM), which alters the wall material properties. We used elastin haploinsufficient (Eln+/-) and wildtype (Eln+/+) mice to investigate how material properties of two different arteries (ascending aorta and carotid artery) change with age, sex, and ECM composition. We used a constitutive model by Dong and Sun that is based on the Holzapfel-Gasser-Ogden (HGO) type, but does not require a discrete number of fibrous ECM families and allows varied deformation coupling. We find that the amount of deformation coupling for the best fit model depends on the artery type. We also find that remodeling to maintain homeostatic (i.e., young, wildtype) values of biomechanical parameters with age, sex, and ECM composition depends on the artery type, with ascending aorta being more adaptable than carotid artery. Fitted material constants indicate sex-dependent remodeling that may be important for determining the time course of arterial stiffening in males and females. We correlated fitted material constants with ECM composition measured by biochemical (ascending aorta) or histological (carotid artery) methods. We show significant correlations between ECM composition and material parameters for the mean values for each group, with biochemical measurements correlating more strongly than histological measurements. Understanding how arterial stiffening depends on age, sex, ECM composition, and artery type may help design effective, personalized clinical treatment strategies.

Elastin recoil is driven by the hydrophobic effect

Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness, and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2 × 109 stretching/contracting cycles without replacement, and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over 50 y, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR, and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with cosolvents. When stretched, elastin's heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when cosolvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism where hardening from crystallization can occur, is the origin of elastin's unusual resilience.

Platelet Proteome Reveals Novel Targets for Hypercoagulation in Pseudoexfoliation Syndrome.

Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of abnormal extracellular matrix material in ocular and non-ocular tissues, including blood vessel walls. Clot-forming dysfunction might be responsible for venous thrombosis in PEX. We investigated global coagulation, the proteome, and functions of platelets in PEX patients and aimed to determine prognostic biomarkers for thrombosis risk in PEX. Peripheral blood was collected from PEX and retinal vein occlusion (RVO) patients, and age-sex matched controls. Viscoelastic hemostasis was evaluated by rotational thromboelastometry (ROTEM). Platelet markers (CD41, CD42, CD61, and CD62p) and endothelial markers (P-selectin, E-selectin, and von Willebrand factor) were investigated by flow cytometry and ELISA, respectively. The platelet proteome was analyzed by 2D fluorescence difference gel electrophoresis followed by mass spectrometry. Clot formation time (CFT) is significantly reduced in PEX patients compared to the controls (p < 0.05). P-selectin levels were higher in PEX patients than in controls (p < 0.05); E-selectin and von Willebrand factor remained unchanged. The monitorization of CFT by ROTEM, and soluble P-selectin, may help assess thrombotic risk in PEX patients. Proteomic analysis revealed differential expression of Profilin-1 in platelets. Profilin-1 regulates the stability of actin-cytoskeleton and may contribute to impaired platelet hemostatic functions. Increased P-selectin levels together with impaired coagulation dynamics might be responsible for the thrombotic events in PEX disease.

AGE-RELATED MECHANISMS OF ALTERED TENDON STRUCTURE AND FUNCTION

During aging, tendons demonstrate substantial disruptions in homeostasis, leading to impairments in structure-function. Impaired tendon function contributes to substantial declines quality of life during aging. Aged tendons are more likely to undergo spontaneous rupture, and the healing response following injury is impaired in aged tendons. Thus, there is a need to develop strategies to maintain tendon homeostasis and healing capacity through the lifespan. Tendon cell density sharply declines by ∼12 months of age in mice, and this low cell density is retained in geriatric tendons. Our data suggests that this decline in cellularity initiates a degenerative cascade due to insufficient production of the extracellular matrix (ECM) components needed to maintain tendon homeostasis. Thus, preventing this decline in tendon cellularity has great potential for maintaining tendon health. Single cell RNA sequencing analysis identifies two changes in the aged tendon cell environment. First, aged tendons primarily lose tenocytes that are associated with ECM biosynthesis functions. Second, the tenocytes that remain in aged tendons have disruptions in proteostasis and an increased pro-inflammatory phenotype, with these changes collectively termed ‘programmatic skewing'. To determine which of these changes drives homeostatic disruption, we developed a model of tenocyte depletion in young animals. This model decreases tendon cellularity to that of an aged tendon, including decreased biosynthetic tenocyte function, while age-related programmatic skewing is absent. Loss of biosynthetic tenocyte function in young tendons was sufficient to induce homeostatic disruption comparable to natural aging, including deficits in ECM organization, composition, and material quality, suggesting loss biosynthetic tenocytes as an initiator of tendon degeneration. In contrast, our data suggest that programmatic skewing underpins impaired healing in aged tendons. Indeed, despite similar declines in the tenocyte environment, middle-aged and young-depleted tendons mount a physiological healing response characterized by robust ECM synthesis and remodeling, while aged tendons heal with insufficient ECM.

Nanobiomaterials in the Management of Oral Biofilms

The oral cavity inhabits diverse microorganisms with members of all age groups including children being associated with oral infections. Bacteria being the principle component of this resident microflora along with the diverse group of other species reflect the wide range of viscerously derived nutrients, the heterogenous habitats for colonization contributing to the survival of the biofilm. Biofilms constitute surface- adherent communities of microorganisms like Streptococcus mutans along with other bacterial species embedded in an extracellular matrix material. The commensal relationship between the host and the microflora can be disrupted in numerous ways, resulting in compromised oral health causing conditions like dental caries and endodontic infections, gingival and periodontal conditions, oral candidiasis and peri-implantitis. Biofilm-related infections prove to be a major threat from both an economical and health perspective. Innovative treatment options remain limited despite of advancements in understanding the mechanisms of biofilm formation. The concept of nanotechnology is based on controlling atoms on an individual basis and creating structures less than 100 nm (10_7 m) in size thereby greatly influencing the properties. Incorporation of metals and other nanoparticles with polymers and coating of surfaces provide antimicrobial and anti-adhesive benefits within the oral cavity. Therefore, this paper seeks to throw light into the current advancements in nanoparticle mediated treatment approaches of oral biofilms and its future implications in pediatric dentistry. Keywords: Biofilm, Oral Cavity, Metal Oxides, Nanoparticles, Reactive Oxygen Species, Antibiofilm Efficacy

Bone Organoids: Recent Advances and Future Challenges.

Bone defects stemming from tumorous growths, traumatic events, and diverse conditions present a profound conundrum in clinical practice and research. While bone has the inherent ability to regenerate, substantial bone anomalies require bone regeneration techniques. Bone organoids represent a new concept in this field, involving the 3D self-assembly of bone-associated stem cells guided in vitro with or without extracellular matrix material, resulting in a tissue that mimics the structural, functional, and genetic properties of native bone tissue. Within the scientific panorama, bone organoids ascend to an esteemed status, securing significant experimental endorsement. Through a synthesis of current literature and pioneering studies, this review offers a comprehensive survey of the bone organoid paradigm, delves into the quintessential architecture and ontogeny of bone, and highlights the latest progress in bone organoid fabrication. Further, existing challenges and prospective directions for future research are identified, advocating for interdisciplinary collaboration to fully harness the potential of this burgeoning domain. Conclusively, as bone organoid technology continues to mature, its implications for both clinical and research landscapes are poised to be profound.

High-strength and high-toughness ECM films with the potential for peripheral nerve repair

Extracellular matrix (ECM) scaffolds are widely applied in the field of regeneration as the result of their irreplaceable biological advantages, and the preparation of ECM scaffolds into ECM hydrogels expands the applications to some extent. However, weak mechanical properties of current ECM materials limit the complete exploitation of ECM’s biological advantages. To enable ECM materials to be utilized in applications requiring high strength, herein, we created a kind of new ECM material, ECM film, and evaluated its mechanical properties. ECM films exhibited outstanding toughness with no cracks after arbitrarily folding and crumpling, and dramatically high strength levels of 86 ± 17.25 MPa, the maximum of which was 115 MPa. Such spectacular high-strength and high-toughness films, containing only pure ECM without any crosslinking agents and other materials, far exceed current pure natural polymer gel films and even many composite gel films and synthetic polymer gel films. In addition, both PC12 cells and Schwann cells cultured on the surface of ECM films, especially Schwann cells, showed good proliferation, and the neurite outgrowth of the PC12 cells was promoted, indicating the application potential of ECM film in peripheral nerve repair.

Comparison of Ultrastructure, Extracellular Matrix, and Drug Susceptibility in M. avium subs. hominissuis Biofilms.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.

Design and Bioprinting of Decellularized Extracellular Matrix-Based Bioinks for Skin Tissue Engineering

3D bioprinting is a powerful enabling technology for the automated fabrication of biomimetic constructs for skin modelling and repair with high resolution and reproducibility. Bioinks, which often comprise the combination of cells and printable biomaterials, are essential for bioprinting 3D cell-laden constructs emulating the architecture, composition and function of native skin. Tissue- and organ-specific decellularized extracellular matrix (dECM) materials are assuming a significant role in bioink design owing their capacity to provide native biophysical and biochemical signals that can elicit distinct cellular responses toward tissue development. Herein, we discuss the rational design of dECM-based bioinks, focusing on processing methods to obtain dECM as well as on bioprinting strategies to engineer 3D constructs for applications in wound healing and in vitro skin modelling.

Decellularized diseased tissues: current state-of-the-art and future directions.

Decellularized matrices derived from diseased tissues/organs have evolved in the most recent years, providing novel research perspectives for understanding disease occurrence and progression and providing accurate pseudo models for developing new disease treatments. Although decellularized matrix maintaining the native composition, ultrastructure, and biomechanical characteristics of extracellular matrix (ECM), alongside intact and perfusable vascular compartments, facilitates the construction of bioengineered organ explants in vitro and promotes angiogenesis and tissue/organ regeneration in vivo, the availability of healthy tissues and organs for the preparation of decellularized ECM materials is limited. In this paper, we review the research advancements in decellularized diseased matrices. Considering that current research focuses on the matrices derived from cancers and fibrotic organs (mainly fibrotic kidney, lungs, and liver), the pathological characterizations and the applications of these diseased matrices are mainly discussed. Additionally, a contrastive analysis between the decellularized diseased matrices and decellularized healthy matrices, along with the development in vitro 3D models, is discussed in this paper. And last, we have provided the challenges and future directions in this review. Deep and comprehensive research on decellularized diseased tissues and organs will promote in-depth exploration of source materials in tissue engineering field, thus providing new ideas for clinical transformation.

Stiffening mechanisms in stochastic athermal fiber networks.

Stochastic athermal networks composed of fibers that deform axially and in bending strain stiffen much faster than thermal networks of axial elements, such as elastomers. Here we investigate the physical origin of stiffening in athermal network materials. To this end, we use models of stochastic networks subjected to uniaxial deformation and identify the emergence of two subnetworks, the stress path subnetwork (SPSN) and the bending support subnetwork (BSSN), which carry most of the axial and bending energies, respectively. The BSSN controls lateral contraction and modulates the organization of the SPSN during deformation. The SPSN is preferentially oriented in the loading direction, while the BSSN's preferential orientation is orthogonal to the SPSN. In nonaffine networks stiffening is exponential, while in close-to-affine networks it is quadratic. The difference is due to a much more modest lateral contraction in the approximately affine case and to a stiffer BSSN. Exponential stiffening emerges from the interplay of the axial and bending deformation modes at the scale of individual or small groups of fibers undergoing large deformations and being subjected to the constraint of rigid cross-links, and it is not necessarily a result of complex interactions involving many connected fibers. An apparent third regime of quadratic stiffening may be evidenced in nonaffinely deforming networks provided the nominal stress is observed. This occurs at large stretches, when the BSSN contribution of stiffening vanishes. However, this regime is not present if the Cauchy stress is used, in which case stiffening is exponential throughout the entire deformation. These results shed light on the physical nature of stiffening in a broad class of materials including connective tissue, the extracellular matrix, nonwovens, felt, and other athermal network materials.

A biomimetic in situ mineralization ECM composite scaffold to promote endogenous bone regeneration

Bone tissue engineering scaffolds constructed from single-component organic materials have inherent limitations. Inspired by the hierarchical structure of physiological natural bone hard tissues, our research explores the construction of organic-inorganic composite scaffold for bone regeneration. In this study, we used a natural and readily obtainable extracellular matrix (ECM) material, i.e., decellularized small intestinal submucosa (SIS), to build the organic component of a phosphorylated hydroxyapatite nanocrystal-containing composite scaffold (nHA@SIS). Guided by polymer-induced liquid-precursor theory, we introduced a soluble inorganic mineralization solution to achieve an inorganic component of nHA@SIS. Using in situ mineralization, we successfully formed inorganic component within SIS and constructed nHA@SIS composite scaffold. We analyzed the physicochemical properties and the osteogenic role of nHA@SIS via a series of in vitro and in vivo studies. Compared with SIS scaffold, the nHA@SIS possessed suitable physicochemical properties, maintained the excellent cell activity of SIS and better guided reorganization of the cell skeleton, thereby achieving superior osteoconductivity and maintaining osteoinductivity at the protein and gene levels. Furthermore, the rat cranial defect area in the nHA@SIS scaffold group was mostly repaired after 12 weeks of implantation, with a larger amount of higher-density new bone tissue being visible at the edge and center than SIS and blank control group. This significantly improved in vivo osteogenic ability indicated the great potential of nHA@SIS for bone tissue engineering applications.

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.

Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes. EVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting. Results showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronectin and EDIL3 were significantly less abundant in glaucomatous EVs (<0.3 fold change across all groups) compared to non-glaucomatous EVs. Overall, these data establish that ECM materials are prominent proteomic cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma.

Biocompatible Human Placental Extracellular Matrix Derived Hydrogels.

Solubilizing extracellular matrix (ECM) materials and transforming them into hydrogels has expanded their potential applications both in vitro and in vivo. In this study, hydrogels are prepared by decellularization of human placental tissue using detergent and enzymes and by the subsequent creation of a homogenized acellular placental tissue powder (P-ECM). A perfusion-based decellularization approach is employed using detergent and enzymes. The P-ECM with and without gamma irradiation is then utilized to prepare P-ECM hydrogels. Physical and biological evaluations are conducted to assess the suitability of the P-ECM hydrogels for biocompatibility. The decellularized tissue has significantly reduced cellular content and retains the major ECM proteins. Increasing the concentration of P-ECM leads to improved mechanical properties of the P-ECM hydrogels. The biocompatibility of the P-ECM hydrogel is demonstrated through cell proliferation and viability assays. Notably, gamma-sterilized P-ECM does not support the formation of a stable hydrogel. Nonetheless, the use of HCl during the digestion process effectively decreases spore growth and bacterial bioburden. The study demonstrates that P-ECM hydrogels exhibit physical and biological attributes conducive to soft tissue reconstruction. These hydrogels establish a favorable microenvironment for cell growth and the need for investigating innovative sterilization methods.

Decellularised hard palate mucosal extracellular matrix material promoting osteogenesis.

Decellularised hard palate mucosal extracellular matrix material promoting osteogenesis.

Mechanical Changes in Fibrous Tissue Exposed to Non‐Convergent Ultrasound

Fibrosis is a disease that involves an abnormal accumulation of extracellular matrix material such as collagen, leading to organ failure. This type of organ failure accounts for around one‐third of all deaths worldwide. However, since there are no drugs which are curative for this disease, the development of non‐invasive treatment strategies is important. In this study, we developed a method of softening fibrotic tissue using non‐converging ultrasound irradiation, a non‐thermal treatment. The generation of cavitation in a standing wave caused the mechanical strength of the fibrotic tissue to decrease in an irradiation time‐dependent manner. The appearance of the fibrous tissue also changed after irradiation, possibly due to the fracture of collagen fibers by ultrasonic cavitation. This method has the potential to treat a wide range of fibrotic tissues. © 2023 Institute of Electrical Engineer of Japan and Wiley Periodicals LLC.

(021) A Novel Model to Study Fibrosis and Scarring in Vulvar Lichen Sclerosus

Abstract Introduction Lichen sclerosus is a debilitating chronic condition where the epidermal layer of the genitals whitens, thickens, and becomes fragile. Lichen sclerosus can be extremely uncomfortable and is accompanied by a wide-range of consequences in biological, psychological, and social domains of life. Patients frequently report intense itching, burning, and pain localized to their genitals, and many experience scarring, bruising, and architectural changes. These symptoms not only limit sexual intercourse, but also impair daily functions, psychological well-being, and interpersonal relationships. Alarmingly, lichen sclerosus can occasionally develop into cancer, however, its etiology remains poorly understood. There is no cure for lichen sclerosus and current treatments only manage symptoms. As a result, we aim to establish an in vitro model of vulvar lichen sclerosus where distinct phenotypic differences between scarred and non-scarred areas can be observed. We aim to use our model to elucidate the molecular mechanisms involved in disease and to identify biomarkers that could be used to identify new targets for therapeutics. Objective To analyze fibroblast-to-myofibroblast transitions and production of extracellular matrix materials in cells derived from scarred vs. non-scarred tissue to improve our understanding of the scarring mechanism implicated in lichen sclerosus. Methods Vulvar punch biopsies collected from scarred and adjacent unaffected areas in anatomically female patients undergoing surgical treatment for lichen sclerosus were used to generate a library of primary fibroblast strains. Cells were treated with transforming growth factor beta (TGF-β) and pro-inflammatory stimuli to investigate scarring and inflammatory pathways, respectively. Fibroblast-to-myofibroblast transitions were assessed using multi-plex fluorescent live cell imaging and western blotting of myofibroblast markers, such as alpha-smooth muscle actin (α-SMA). Production of extracellular matrix proteins such as collagen, fibronectin, and calponin was also analyzed via western blotting. Results Morphological analysis of our collection of lichen sclerosus fibroblasts revealed that cells cultured from scarred areas tend to be large and irregularly shaped, resembling myofibroblasts. In contrast, cells from unaffected areas were observed to be thin and spindle-shaped, resembling fibroblasts. TGF-β treatment resulted in scar fibroblasts significantly upregulating expression of α-SMA, whereas this trend was not observed in controls. Our preliminary data suggests that TGF-β treatment may increase the expression of additional matrix proteins, such as collagen. This is an area of active research we are continuing to investigate. Conclusions Lichen sclerosus is a severely detrimental condition that is poorly characterized. Our results suggest that scarring pathways, such as TGF-β signaling cascades, may be implicated in the mechanism. Fibroblasts from scarred areas more readily undergo fibroblast-to-myofibroblast transitions, which may result in accumulation of extracellular matrix materials and fibrosis is lichen sclerosus. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: SPM Therapeutics.

A native extracellular matrix material for tissue engineering applications: Characterization of pericardial fluid.

Tissue engineering applications are widely used to repair and regenerate damaged tissues and organs. A scaffold, which is an important component in tissue engineering, provides a 3D environment for cells. In this study, the usability of PF components for the production of an ideal scaffold was investigated. For this aim, pericardial fluid (PF) was harvested from the bovine heart, then its structure and components were characterized. The results of Raman spectroscopy analysis, histological staining, and scanning electron microscopy (SEM) shows that the pericardial fluid contains collagen type I and IV, elastin, fibrin, and glycosaminoglycan (GAG), which are native extracellular matrix (ECM) components. The results demonstrated that (i) PF contains native ECM proteins and GAG such as collagen types I, III, and IV, elastin, and fibrin. (ii) The PF is highly similar to the native ECM structure. (iii) PF can significantly contribute to many tissue engineering studies as a native ECM material to increase the biocompatibility of biomaterials and to several in vitro/in vivo cell culture studies. (iv) PF containing multiple ECM molecules, can be used alone or together with hyaluronic acid, poly(ethylene glycol) (PEG), alginate, chitosan, matrigel, and gelatin methacryloyl (GelMA) materials in bioprinting systems for eliminating the disadvantages of these materials.

Endosidin 5 disruption of the Golgi Apparatus and extracellular matrix secretion in the unicellular charophyte, Penium margaritaceum.

Endosidins are a group of small molecular weight compounds, first identified by "chemical biology" screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate effects of Endosidin 5 (ES5) on the Golgi Apparatus and the secretion of extracellular matrix (ECM) components, Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum's extensive Golgi Apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi Apparatus and secretion of ECM material caused by Endosidin 5. Changes to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy, and transmission electron microscopy were used to assess changes to the Golgi Apparatus, the cell wall and the vesicular network. Electron tomography was also performed in order to detail the changes to the Golgi Apparatus. While other endosidins were able to impact EPS secretion and cell wall expansion only ES5 completely inhibited EPS secretion and cell wall expansion over 24h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way than other endomembrane inhibitors such as Brefeldin A and Concanamycin A.