closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition

The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.     

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca²⁺-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).     

Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Micro 3D cell culture systems for cellular behavior studies: Culture matrices,devices, substrates,and in‐situ sensing methods

Microfabricated systems equipped with 3D cell culture devices and in‐situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio‐functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near‐cell surface detection of excreted cellular compounds, SERS‐based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development.     

Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits

Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids, therefore leaving the possibility of rapid evolution through gametic interaction wide open. At this point, however, our results support previous suggestions that divergences in ecology, particularly, ovipositional substrate divergences may be a strong force driving the evolution of eggshell proteins.     

Role of surface chemistry in protein remodeling at the cell-material interface

Background

The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM) proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation.

Methodology/Principal Findings

This work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs) of –OH and –CH₃ terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN) was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way.

Conclusions/Significance

This work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell-material interface, which gains a distinct bioengineering interest as a new tool to trigger matrix degradation in different biomedical applications.     

The Stiffness of Three-dimensional Ionic Self-assembling Peptide Gels Affects the Extent of Capillary-like Network Formation

Improving our ability to control capillary morphogenesis has implications for not only better understanding of basic biology, but also for applications in tissue engineering and in vitro testing. Numerous biomaterials have been investigated as cellular supports for these applications and the biophysical environment biomaterials provide to cells has been increasingly recognized as an important factor in directing cell function. Here, the ability of ionic self-assembling peptide gels to support capillary morphogenesis and the effect of their mechanical properties is investigated. When placed in a physiological salt solution, these oligopeptides spontaneously self-assemble into gels with an extracellular matrix (ECM)-like microarchitecture. To evaluate the ability of three-dimensional (3D) self-assembled peptide gels to support capillary-like network formation, human umbilical vein endothelial cells (HUVECs) were embedded within RAD16-I ((RADA)₄) or RAD16-II ((RARADADA)₂) peptide gels with various stiffness values. As peptide stiffness is decreased cells show increased elongation and are increasingly able to contract gels. The observation that capillary morphogenesis is favored in more malleable substrates is consistent with previous reports using natural biomaterials. The structural properties of peptide gels and their ability to support capillary morphogenesis in vitro make them promising biomaterials to investigate for numerous biomedical applications.     

Arteriolar vascular smooth muscle cells: mechanotransducers in a complex environment

Contraction of small artery (diameters typically less than 250 μm) vascular smooth muscle cells (VSMCs) plays a critical role in local control of blood flow and arterial pressure through its affect on vascular caliber. Specifically, contraction of small arteries in response to increased intraluminal pressure is referred to as the myogenic response and represents an important role for mechanotransduction. Critical questions remain as to how changes in pressure are sensed by VSMCs and transduced across the cell membrane to tune the contractile state of the cell. Recent studies suggest a pivotal role for interactions between VSMCs and extracellular matrix (ECM) proteins. Thus, pressure-induced deformation of ECM proteins and their cell surface receptors (for example, integrins) may initiate contraction and cytoskeletal remodeling through modulation of ion channels, membrane depolarization, increased intracellular Ca(2+) and actomyosin crossbridge cycling. Importantly, it is argued that the contractile properties of small artery VSMCs reflect an intimate and integrated interaction with their extracellular environment and the three-dimensional structure of the vessel wall.     

Anchorless adhesins and invasins of Gram-positive bacteria: a new class of virulence factors

Bacterial adherence to and invasion of eukaryotic cells are important mechanisms of pathogenicity. Most Gram-positive bacteria interact with the components of the host extracellular matrix (ECM) to adhere to, colonize and invade cells and tissues. The bacterial proteins that bind to components of the ECM harbour signal sequences for their secretion and mechanisms of anchoring to the host cell surface. However, in recent years, some cell-surface adhesins and invasins of Gram-positive bacteria have been described that do not possess a signal sequence or a membrane anchor. These proteins are secreted by an as-yet-unknown mechanism and are probably localized on the bacterial surface by reassociation. These anchorless but surface-located adhesins and invasins represent a new class of virulence factors.     

Bacterial adhesion to animal tissues: protein determinants for recognition of extracellular matrix components

The extracellular matrix (ECM) is present within all animal tissues and organs. Actually, it surrounds the eukaryotic cells composing the four basic tissue types, i.e. epithelial, muscle, nerve and connective. ECM does not solely refer to connective tissue but composes all tissues where its composition, structure and organization vary from one tissue to another. Constituted of the four main fibrous proteins, i.e. collagen, fibronectin, laminin and elastin, ECM components form a highly structured and functional network via specific interactions. From the basement membrane to interstitial matrix, further heterogeneity exists in the organization of the ECM in various tissues and organs also depending on their physiological state. Back to a molecular level, bacterial proteins represent the most significant part of the microbial surface components recognizing adhesive matrix molecules (MSCRAMM). These cell surface proteins are secreted and localized differently in monoderm and diderm–LPS bacteria. While one collagen‐binding domain (CBD) and different fibronectin‐binding domains (FBD1 to 8) have been registered in databases, much remains to be learned on specific binding to other ECM proteins via single or supramolecular protein structures. Besides theinteraction of bacterial proteins with individual ECM components, this review aims at stressing the importance of fully considering the ECM at supramolecular, cellular, tissue and organ levels. This conceptual view should not be overlooked to rigorously comprehend the physiology of bacterial interaction from commensal to pathogenic species.     

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.     

Structure of shells of eggs of Callisaurus draconoides (Reptilia, Squamata, Iguanidae)

Female zebra-tailed lizards (Iguanidae: Callisaurus draconoides ) lay roughly ovoid eggs with thin, highly extensible shells. The outer surface of the eggshell is a thin, calcareous crust of calcium carbonate in the calcite morph. Immediately beneath the crystalline matrix is a shell membrane composed of multiple layers of fibres organized into an undulating series of troughs and crests, apparent in both cross-section and surface view. The outer surface of the shell membrane is differentiated into a tightly woven fibrous mat that may serve to anchor the calcareous layer to the membrane. Organization of the eggshell into a series of troughs and crests serves to increase the surface area available for contact with the substrate and, presumably, to increase the capacity of the eggshell to stretch as the egg absorbs water.     

Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.     

Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces

Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.     

中国特产雉类—褐马鸡,藏马鸡和蓝马鸡卵壳的电镜观察

迄今为止,在国内有关野生鸟类卵壳的超微结构报道尚少。我们对中国特产鸡类——三种马鸡卵壳的表皮、气孔、木栅层、锥体层、乳头结、壳膜和基帽进行了扫描巨镜观察,并对结果进行了比较、讨论。认为三种马鸡不同结构的形态,对研究它们的亲缘关系提供了新的价值。     

The eggshell in the C. elegans oocyte-to-embryo transition

In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development.     

Integrin receptors: the dynamic modulators of endometrial function

Cell-cell and cell-extracellular matrix (ECM) interactions play a critical role in various developmental processes, including differentiation, proliferation and migration of cells. ECM proteins can influence cellular function thus creating a complex feedback mechanism. The adhesion of cells to each other, their ECM proteins and endothelial surfaces is mediated by a variety of membrane proteins collectively known as adhesion molecules. Adhesion molecules have been further divided into five subfamilies, the integrins, the selectins, the cadherins, the mucins and the immunoglobulin superfamily. Members of the integrin family of cell surface adhesion receptors are important mediators of cell-ECM contact. Integrin receptors are alpha beta heterodimers with a transmembrane segment, a short cytoplasmic domain and a large extracellular domain. The role of integrins in reproduction has been established. Several reasons make these molecules very attractive due to their constant involvement from egg to birth. They participate in sperm-egg interaction, fertilization, implantation and placentation in many species including humans. Integrins provide signals to individual cells essential for growth and development of different tissues. In the present review, we describe (1) the regulatory pathways for controlling expression of integrins in the endometrium, (2) various biomarkers and their role in endometrial function, (3) reproductive disorders in women related to aberrant integrin expression in the endometrium and (4) the functional significance of integrins available from gene knockout studies.     

蛋壳内膜分解菌所产蛋白酶的纯化及其N-末端氨基酸序列测定

从土壤中分离得到的1株产蛋壳内膜分解酶(ESM protease)的铜绿假单胞菌(Pseudomonasaeruginosa).通过对其发酵液进行饱和硫酸铵盐析,二次离子交换层析得到蛋壳内膜分解活性达到304.5 U/mg的目标蛋白,SDS-PAGE电泳显示该酶分子量约为32 kD,通过测定其N-末端15个氨基酸残基为:Ala、Glu、Ala、Gly、Gly、Val、Ala、Gly、Lys、Glu、Asp、Ala、Ala、Glu和Leu.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.