The morphogenesis of the chick primary corneal stroma. I. New observations on collagen organization in vivo help explain stromal deposition and growth

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 135-145
Author(s):  
J.B. Bard ◽  
M.K. Bansal

The primary stroma of the avian cornea contains collagen fibrils in orthogonal array. While investigating the processes underlying its morphogenesis, we have found that stromal organization is not as expected in three important respects. First, the fibrils are not uniform: those near the epithelium (newly laid down) have a maximum diameter of about 20 nm (mean: 17.7 nm), while those near the endothelium (laid down for approx. 40 h) have diameters up to 40 nm (mean: 22.8 nm). Fibrils thus grow rapidly to 20 nm and then continue to enlarge slowly, presumably by diffusion of collagen molecules from the epithelium. Second, the collagen, although orthogonally organized, does not contain layers of parallel fibrils. Instead, SEM observation shows that only a few fibrils lie in a parallel array before this short-range order is broken by orthogonal fibrils in the same plane. Furthermore, fibrils in corneas that had been freeze dried but not critical-point dried for SEM were widely spaced and the intervening gaps were filled by an extensive matrix that was probably composed of the proteoglycans known to be in the stroma. Third, we have shown experimentally that the stromal undulations seen in sections are not present in vivo but are shrinkage artifacts: the less corneas were shrunk for SEM preparation, the less pronounced were the stromal undulations. We also noted that, even after the distortions required for the stroma to undulate, the constituent fibrils remained orthogonally organized. These results give insight into the mechanisms underlying stromal morphogenesis and growth. The observations on the growth of collagen fibrils and on collagen organization show that stromal deposition is a more stochastic process than previously thought and, hence, provides support for the view that a complex self-assembly mechanism underlies both fibrillogenesis and the generation of orthogonal organization. The experiments on, and the analysis of, stromal folding show that fibrils slide over one another as undulations form, with the extensive matrix of hydrated proteoglycans being the likely lubricant. This fluidity of the stromal components probably explains how growth can occur without the structure being distorted.

Development ◽  
1988 ◽  
Vol 103 (Supplement) ◽  
pp. 195-205
Author(s):  
J. B. L. Bard ◽  
M. K. Bansal ◽  
A. S. A. Ross

This paper examines the role of the extracellular matrix (ECM) in the development of the cornea. After a brief summary of the corneal structure and ECM, we describe evidence suggesting that the differentiation of neural crest (NC) cells into endothelium and fibroblasts is under the control of ocular ECM. We then examine the role of collagen I in stromal morphogenesis by comparing normal corneas with those of homozygous Movl3 mice which do not make collagen I. We report that, in spite of this absence, the cellular morphology of the Movl3 eye is indistinguishable from that of the wild type. In the 16-day mutant stroma, however, the remaining collagens form small amounts of disorganized, thin fibrils rather than orthogonally organized 20 nm-diameter fibrils; a result implying that collagen I plays only a structural role and that its absence is not compensated for. It also suggests that, because these remaining collagens will not form the normal fibrils that they will in vitro, fibrillogenesis in the corneal stroma differs from that elsewhere. The latter part of the paper describes our current work on chick stromal deposition using corneal epithelia isolated with an intact basal lamina that lay down in vitro ∼3μm-thick stromas of organized fibrils similar to that seen in vivo. This experimental system has yielded two unexpected results. First, the amount of collagen and proteoglycans produced by such epithelia is not dependent on whether its substratum is collagenous and we therefore conclude that stromal production by the intact epithelium is more autonomous than hitherto thought. Second, chondroitin sulphate (CS), the predominant proteoglycan, appears to play no role in stromal morphogenesis: epithelia cultured in testicular hyaluronidase, which degrades CS, lay down stromas whose organization and fibrildiameter distribution are indistinguishable from controls. One possible role for CS, however, is as a lubricant which facilitates corneal growth: it could allow fibrils to move over one another without deforming their orthogonal organization. Finally, we have examined the processes of fibrillogenesis in the corneal stroma and conclude that they are different from those elsewhere in the embryo and in vitro, perhaps because there is in the primary stroma an unidentified, highly hydrated ECM macromolecule that embeds the fibrils and that may mediate their morphogenesis.


2018 ◽  
Author(s):  
Roxanne Diaz ◽  
Aurore Sanchez ◽  
Jérôme Rech ◽  
Delphine Labourdette ◽  
Jérôme Dorignac ◽  
...  

SummaryChromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS-bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the plasmid F partition system. We found that ‘Nucleation & caging’ is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) does not directly involve ParA, (ii) results in a dynamic structure of discrete size independent of ParB concentration, and (iii) is not perturbed by active transcription but is by protein complexes. We refined the ‘Nucleation & Caging’ model and successfully applied it to the chromosomally-encoded Par system of Vibrio cholerae, indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes.


Author(s):  
Nima Saeidi ◽  
Jeffrey W. Ruberti

Load-bearing tissues owe their mechanical properties to the presence of highly-organized arrays of collagen fibrils. Aligned lamellae in cornea and aligned fascicles in tendon are the best examples of collagen fibrillar organization at the macroscopic level. The process by which collagen is organized in the extracellular matrix (ECM) is still unclear. But it is generally thought to be facilitated locally via “fibripositors” or cell surface “crypts”. According to this theory, fibroblasts create bounded “compartments” in the ECM through which they deposit organized groups of fibrils (in the form of lamellae in the cornea and in the form of fascicles in the tendon) [1, 2]. An alternative hypothesis proposed by Marie Giraud-Guille suggests that fibroblasts concentrate collagen monomers to form cholesteric liquid crystalline patterns that resemble those found in collagenous matrices in vivo [3–8]. Such organization has been demonstrated in vitro using extracted collagen monomers. However, the data presented in these studies focuses principally on the alignment of the collagen molecules and not on the organization and resulting morphology of condensed collagen fibrils. Considering that matrix mechanical properties in vivo are the result of the fibrillar alignment and not the alignment of individual molecules, further investigation of cholesterically organized condensed fibrils and their morphology is necessary.


MRS Advances ◽  
2020 ◽  
Vol 5 (46-47) ◽  
pp. 2401-2407
Author(s):  
Michael Y. Yitayew ◽  
Maryam Tabrizian

AbstractHollow microcapsules prepared via layer-by-layer (LbL) self-assembled polyelectrolytes are prevalent biomaterials in the synthesis of biocompatible delivery systems for drugs, imaging probes, and other macromolecules to control biodistribution and lower toxicity in vivo. The use of LbL self-assembly for the synthesis of these capsules provides several benefits including ease of fabrication, abundance in choice of substrates and coating material, as well as application-specific tunability. This study explores the development of hollow microcapsules by LbL assembly of chitosan and alginate onto live E. coli cells, and also provides a proof-of-concept of this capsule as a delivery platform through the encapsulation of quantum dots as a cargo. The study found that robust bilayers of chitosan/alginate can be formed onto the core substrate (E. coli) containing quantum dots as demonstrated with zeta potential analysis. Confocal microscopy was used to verify cell viability and the internalization of quantum dots into the cells as well as confirmation of the coating using fluorescein-labelled chitosan. Furthermore, transmission electron microscopy (TEM) was used to analyse cells coated with four-bilayers and showed a uniform coating morphology with a capsule thickness of 10-20 nm, which increased to 20-50 nm for hollow capsules after cell lysis. Quantum dot retention in the capsules was demonstrated using fluorescence measurements. Overall, the study shows promising results of a novel fabrication method for hollow microcapsules that uses biocompatible polymers and mild core dissolution conditions using cell templates with applications in sustained release of therapeutics and imaging probes.


2008 ◽  
Vol 58 ◽  
pp. 60-65
Author(s):  
Mitsuyo Okamoto ◽  
E. Iwai ◽  
H. Hatta ◽  
Hitoshi Kohri ◽  
Ichiro Shiota

In bio-systems, nano-composites with complex micro-structures are formed by self-assembly only using low energy at room temperature. If these mechanisms of biological tissue are identified, we can possibly propose a new process to fabricate composites by mimicking tissue formation in vivo. As a bio-material, we paid attention to bio-tissue reinforced with collagen fibrils. Collagen fibrils are of baculiform; Thus the self-assembly process through liquid crystalline transition has been proposed by a French group [1]. In the present study, factors controlling liquid crystalline transition, e.g. concentration and pH, are discussed using collagen solution. When liquid crystalline phase is produced, aligned molecules exhibits optical anisotropy. This anisotropy was observed with a polarized optical microscopy (POM). By observations with POM, development of cholesteric phase in collagen solution was clarified.


1993 ◽  
Vol 105 (4) ◽  
pp. 1045-1055 ◽  
Author(s):  
J.B. Bard ◽  
D.J. Hulmes ◽  
I.F. Purdom ◽  
A.S. Ross

In vivo, the embryonic chick corneal epithelium lays down a stroma of collagen and proteoglycans whose fibrils are unusual as their diameter distribution peaks sharply about a mean of 20 nm. Such epithelia cultured on Nuclepore filters will also lay down a stroma containing 20 nm diameter fibrils, although there is only limited orthogonal organisation. We report here that collagen fibril morphology is critically dependent on the pH of the medium in which the corneal epithelium is cultured and that normal 20 nm diameter fibrils only assemble in a narrow band around neutral pH (approx. 6.9-7.4). At higher pH (7.6-8.1), fibrils in the distal region of the stroma more closely resemble those seen in non-corneal stroma as their diameters can be up to 200 nm even though fibrils near the basal lamina are only about 10 nm in diameter. At low pH (approx. 6.5), there are again wide fibrils, but with the hieroglyphic cross-sections typical of those seen in heritable disorders of N-terminal procollagen processing. Biochemical analysis by SDS-PAGE and fluorography confirms that N-terminal procollagen processing is deficient at this pH. At very low pH (approx. 5.8-6.2), there is little processing of procollagen and the stroma comprises filamentous material with the occasional banded structures typical of those formed by unprocessed procollagen at high concentration. Gel electrophoresis and peptide mapping showed that the collagens produced by the corneal epithelium of the primary stroma included types I, II and V and that total collagen production, as assessed by incorporation of [3H]proline, increased with pH, although the relative amounts of the different collagens produced remained essentially unchanged. While the biochemical data can account for the altered morphologies in the pH range 5.8 to 7.0, the sensitivity of fibril diameter to small changes around neutral pH remains unexplained, but points to the subtle, charge-based interactions necessary for the formation of 20 nm diameter fibrils in the developing cornea.


2007 ◽  
Vol 342-343 ◽  
pp. 929-932
Author(s):  
Qing Rong Wei ◽  
Xiu Dong Yang ◽  
Jian Lu ◽  
Bo Zhang ◽  
Bo Jiang ◽  
...  

As a natural biomaterial, collagen especially pepsin-solubilized collagen (type I) has been used widely in biomedical fields due to its excellent biocompatibility. In this preliminary study, we investigate the effect of some inorganic ions which are frequently utilized in the preparation of collagen on the morphology and crystallinity of fibrils. The scanning electron microscope and x-ray diffraction were applied to analyze the morphology and the crystallization of the reconstituted collagen fibrils, respectively. Although further studies are needed, these initial results indicate that by controlling the self-assembly conditions of collagen molecules, we may achieve the desired properties of fibrillar collagen products.


Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.


Author(s):  
George C. Ruben ◽  
William Krakow

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.


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