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Collagen Fibril Orientation and Corneal Curvature
Structure, Vol. 12, 169–174, February, 2004, 2004 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/j.str.2004.01.019
Previews
Collagen Fibril Orientation and Corneal Curvature
This issue of Structure presents a report (Aghamohammadzadeh et al., 2004) that maps collagen fibril orientation throughout the cornea and explains how this information can be used to understand why the cornea is curved. The paper by Meek’s group (Aghamohammadzadeh et al., 2004) provides a remarkable insight into one of the most poorly understood properties of the cornea, namely how it manages to combine precise curvature with almost perfect transparency. The curvature of the cornea is responsible for two thirds of the refraction of light in the eye and the slightest imperfection in its shape results in astigmatism and refractive error. The cornea consists of an outer epithelial layer and an inner endothelial layer, but it is the central stroma, making up 90% of the thickness of the cornea, which gives it its strength and shape. The stroma is composed of collagen fibrils of a uniform diameter, embedded in a highly hydrated matrix made up mostly of proteoglycans that are implicated in the control of fibril organization (Quantock et al., 2001; Meek et al., 2003). The fibrils are remarkable in the uniformity of their diameters and the regularity of their spacing (Figure 1). Individual fibrils consist of mainly type I collagen but also include type V collagen, which controls fibril diameter (Birk, 2001), and are arranged in layers or lamellae (Figure 1) of various widths, similar to the different layers of wood in a sheet of plywood. Within individual lamella, fibrils lie parallel to each other but at approximately right angles to those in adjacent lamellae (Figure 1). The central cornea contains between 200 and 400 lamellae, and it is implicit that lamellar organization and distribution must control corneal shape and curvature. However, prior to this paper, little or no information existed on the gross orientation and distribution of the lamellae in the cornea and limbus. David Maurice pioneered the use of X-rays to examine the collagen fibril arrangement in the cornea in the 1950s. His early experiments showed that the collagen fibrils were arranged in a lattice, and he formulated the “destructive interference” theory of transparency. Basically, this theory states that when light is scattered by the fibrils in the stroma, their regular arrangement results in destructive interference of light scattered at all angles except for the forward direction (Maurice, 1957), making the material transparent. Although this theory explains the physical principles behind transparency, further investigation of the orientation of collagen fibrils within the cornea and sclera has been largely neglected. Theoretically, electron microscopy (Figure 1) could be used to visualize and map fibril orientation; however, practically this has not proved feasible.
Although fiber X-ray diffraction has previously been used to study fibril diameter and interfibrillar spacing, it is only recently that Meek’s group developed a method for mapping fibril orientation. Work conducted over the last few years has resulted in increasingly sensitive and sophisticated analysis of fibril arrangement in the corneal stroma (Daxer and Fratzl, 1997; Newton and Meek 1998; Meek and Quantock, 2001), culminating in this present study. Here, Meek’s group describe how they used the latest developments in synchrotron X-ray diffraction instrumentation and analysis, collecting over a thousand diffraction patterns from the cornea and limbus under near physiological conditions. By analyzing the primary reflection derived from the lateral spacing of the collagen molecules within the individual fibrils, the group has been able to determine the overall preferred orientation of the collagen fibers throughout the entire cornea and limbus. This data is displayed as polar plots and, also, for the first time ever, as a 2D map, showing that that the arrangement of collagen and lamellae in the cornea and limbus is highly complex. The group reports that there is a preferred orientation of collagen fibrils in the horizontal and vertical directions within the central region of the cornea—this central (optical) region of the cornea has a relatively uniform distribution of preferentially aligned collagen fibrils. Surrounding the central region in a diamond-like arrangement, a set of
Figure 1. Transmission Electron Micrograph Showing the Orientation of Collagen Fibrils in Adjacent Lamellae in the Corneal Stroma The micrograph shows three lamellae from the central corneal stroma. In the middle lamella, the collagen fibers are in cross-section (running toward the reader) and can be seen to be of regular diameter and spacing. In the top and bottom lamellae, the collagen fibers are in longitudinal section (running from side to side) and are at approximate right angles to the collagen fibers in the middle lamella. Scale bar ⫽ 200 nm.
Structure 170
curved anchoring lamella is apparent to the researchers, but perhaps most interesting of all is the observation that an annulus of collagen fibers encircle the limbus. Meek and colleagues suggest that the change in orientation of the fibrils as they move toward the limbus accounts for the flattening of curvature of the cornea in this region, and their finding of the circumferential ring of fibrils at the limbus also provides a very elegant explanation as to how the transition in curvature between the cornea and sclera is achieved. It is difficult to overemphasize the importance of this work. The majority of corneal diseases result from disruption to the fibril arrangement in the corneal stroma. Most individuals will develop a refractive error at some point in their lives, and the use of corrective refractive surgery is ever more popular and may well become the norm in the future. The current leading corrective procedures include photorefractive keratectomy and laser in situ keratomileusis, both of which involve the ablation of corneal lamellae from the stroma. To date, these technologies have been improved and developed using a fairly empirical approach, largely because of the lack of information available on the orientation of collagen fibrils in the cornea. Consequently, to some extent, corneal surgery is unpredictable and can result in over or under correction, or in the development of astigmatism (Melki and Azar, 2001). This key study should allow corneal surgeons to more accurately predict the outcome of incisions or ablations on different regions of the cornea. Finally, fiber X-ray diffraction has always been consid-
ered much less glamorous and important than protein crystallography. Extracting information from the relatively diffuse reflections arising from systems with shortrange order is difficult and time-consuming work. However, as this paper shows, with perseverance immensely useful information can be obtained. Hopefully, the publication of this paper will lead to a wider appreciation and application of this approach. Nigel J. Fullwood Biological Sciences, IENS Lancaster University Lancaster LA1 4YQ United Kingdom Selected Reading Aghamohammadzadeh, H., Newton, R.H., and Meek, K.M. (2004). Structure 12, this issue, 249–256. Birk, D.E. (2001). Micron 32, 223–237. Daxer, A., and Fratzl, P. (1997). Invest. Ophthalmol. Vis. Sci. 38, 121–129. Quantock, A.J., Meek, K.M., and Chakravarti, S. (2001). Invest. Ophthalmol. Vis. Sci. 42, 1750–1756. Maurice, D.M. (1957). J. Physiol. 136, 263–286. Meek, K.M., and Quantock, A.J. (2001). Prog. Ret. Eye Res. 20, 95–137. Meek, K.M., Quantock, A.J., Boote, C., Liu, C.Y., and Kao, W.W. (2003). Matrix Biol. 22, 467–475. Melki, S.A., and Azar, D.T. (2001). Surv. Ophthalmol. 46, 95–116. Newton, R.H., and Meek, K.M. (1998). Biophys. J. 75, 2508–2512.
Structure, Vol. 12, Feburary, 2004, 2004 Elsevier Science Ltd. All rights reserved.
Accessing Information on the Conformational Flexibility of Molecular Machines Acquiring information to describe the conformational flexibility of molecular machines is necessary to understand their mechanics but is technically difficult to achieve. In this issue of Structure, Chiu and colleagues effectively tackle this problem using 3D cryo-electron microscopy with impressive results.
The Problem At the heart of most biological processes, a molecular machine performs a given function or range of functions, generally by orchestrating a range of interactions with different cofactors. Molecular machines, the orchestra conductors, need to be physically flexible to accommodate all the necessary interactions and so must exhibit a certain degree of polymorphism. Obviously, a detailed
DOI 10.1016/j.str.2004.01.017
structural analysis of this polymorphism would shed much-needed light on the molecular mechanism that facilitates execution of a particular function. But how are these flexible intermediate structures to be analyzed and studied? More precisely, what experimental techniques can capture an image of a megadalton flexible complex and reveal its three-dimensional structure? The answer: certainly not many. In fact, three-dimensional cryo-electron microscopy (3D-cryoEM) is currently the best technique available to access this information experimentally since it does not require specimens to be arranged in a periodic manner, which would be difficult to achieve with mixtures of conformations, and it can deal with very large complexes. However, common image-processing approaches in the field do not work well if the sample is polymorphic. Obviously, for 3D-cryoEM to deliver, the analytical methods have to be improved, which is precisely the topic of Chiu and coworkers’ contribution published in this issue (Brink et al., 2004). The Prior Context in the Field of 3D-cryoEM A number of approaches to add the “flexibility” dimension to 3D-cryoEM studies have been considered. One approach directly works at the level of sample prepara-
DOI 10.1016/j.str.2004.01.019
Previews
Collagen Fibril Orientation and Corneal Curvature
This issue of Structure presents a report (Aghamohammadzadeh et al., 2004) that maps collagen fibril orientation throughout the cornea and explains how this information can be used to understand why the cornea is curved. The paper by Meek’s group (Aghamohammadzadeh et al., 2004) provides a remarkable insight into one of the most poorly understood properties of the cornea, namely how it manages to combine precise curvature with almost perfect transparency. The curvature of the cornea is responsible for two thirds of the refraction of light in the eye and the slightest imperfection in its shape results in astigmatism and refractive error. The cornea consists of an outer epithelial layer and an inner endothelial layer, but it is the central stroma, making up 90% of the thickness of the cornea, which gives it its strength and shape. The stroma is composed of collagen fibrils of a uniform diameter, embedded in a highly hydrated matrix made up mostly of proteoglycans that are implicated in the control of fibril organization (Quantock et al., 2001; Meek et al., 2003). The fibrils are remarkable in the uniformity of their diameters and the regularity of their spacing (Figure 1). Individual fibrils consist of mainly type I collagen but also include type V collagen, which controls fibril diameter (Birk, 2001), and are arranged in layers or lamellae (Figure 1) of various widths, similar to the different layers of wood in a sheet of plywood. Within individual lamella, fibrils lie parallel to each other but at approximately right angles to those in adjacent lamellae (Figure 1). The central cornea contains between 200 and 400 lamellae, and it is implicit that lamellar organization and distribution must control corneal shape and curvature. However, prior to this paper, little or no information existed on the gross orientation and distribution of the lamellae in the cornea and limbus. David Maurice pioneered the use of X-rays to examine the collagen fibril arrangement in the cornea in the 1950s. His early experiments showed that the collagen fibrils were arranged in a lattice, and he formulated the “destructive interference” theory of transparency. Basically, this theory states that when light is scattered by the fibrils in the stroma, their regular arrangement results in destructive interference of light scattered at all angles except for the forward direction (Maurice, 1957), making the material transparent. Although this theory explains the physical principles behind transparency, further investigation of the orientation of collagen fibrils within the cornea and sclera has been largely neglected. Theoretically, electron microscopy (Figure 1) could be used to visualize and map fibril orientation; however, practically this has not proved feasible.
Although fiber X-ray diffraction has previously been used to study fibril diameter and interfibrillar spacing, it is only recently that Meek’s group developed a method for mapping fibril orientation. Work conducted over the last few years has resulted in increasingly sensitive and sophisticated analysis of fibril arrangement in the corneal stroma (Daxer and Fratzl, 1997; Newton and Meek 1998; Meek and Quantock, 2001), culminating in this present study. Here, Meek’s group describe how they used the latest developments in synchrotron X-ray diffraction instrumentation and analysis, collecting over a thousand diffraction patterns from the cornea and limbus under near physiological conditions. By analyzing the primary reflection derived from the lateral spacing of the collagen molecules within the individual fibrils, the group has been able to determine the overall preferred orientation of the collagen fibers throughout the entire cornea and limbus. This data is displayed as polar plots and, also, for the first time ever, as a 2D map, showing that that the arrangement of collagen and lamellae in the cornea and limbus is highly complex. The group reports that there is a preferred orientation of collagen fibrils in the horizontal and vertical directions within the central region of the cornea—this central (optical) region of the cornea has a relatively uniform distribution of preferentially aligned collagen fibrils. Surrounding the central region in a diamond-like arrangement, a set of
Figure 1. Transmission Electron Micrograph Showing the Orientation of Collagen Fibrils in Adjacent Lamellae in the Corneal Stroma The micrograph shows three lamellae from the central corneal stroma. In the middle lamella, the collagen fibers are in cross-section (running toward the reader) and can be seen to be of regular diameter and spacing. In the top and bottom lamellae, the collagen fibers are in longitudinal section (running from side to side) and are at approximate right angles to the collagen fibers in the middle lamella. Scale bar ⫽ 200 nm.
Structure 170
curved anchoring lamella is apparent to the researchers, but perhaps most interesting of all is the observation that an annulus of collagen fibers encircle the limbus. Meek and colleagues suggest that the change in orientation of the fibrils as they move toward the limbus accounts for the flattening of curvature of the cornea in this region, and their finding of the circumferential ring of fibrils at the limbus also provides a very elegant explanation as to how the transition in curvature between the cornea and sclera is achieved. It is difficult to overemphasize the importance of this work. The majority of corneal diseases result from disruption to the fibril arrangement in the corneal stroma. Most individuals will develop a refractive error at some point in their lives, and the use of corrective refractive surgery is ever more popular and may well become the norm in the future. The current leading corrective procedures include photorefractive keratectomy and laser in situ keratomileusis, both of which involve the ablation of corneal lamellae from the stroma. To date, these technologies have been improved and developed using a fairly empirical approach, largely because of the lack of information available on the orientation of collagen fibrils in the cornea. Consequently, to some extent, corneal surgery is unpredictable and can result in over or under correction, or in the development of astigmatism (Melki and Azar, 2001). This key study should allow corneal surgeons to more accurately predict the outcome of incisions or ablations on different regions of the cornea. Finally, fiber X-ray diffraction has always been consid-
ered much less glamorous and important than protein crystallography. Extracting information from the relatively diffuse reflections arising from systems with shortrange order is difficult and time-consuming work. However, as this paper shows, with perseverance immensely useful information can be obtained. Hopefully, the publication of this paper will lead to a wider appreciation and application of this approach. Nigel J. Fullwood Biological Sciences, IENS Lancaster University Lancaster LA1 4YQ United Kingdom Selected Reading Aghamohammadzadeh, H., Newton, R.H., and Meek, K.M. (2004). Structure 12, this issue, 249–256. Birk, D.E. (2001). Micron 32, 223–237. Daxer, A., and Fratzl, P. (1997). Invest. Ophthalmol. Vis. Sci. 38, 121–129. Quantock, A.J., Meek, K.M., and Chakravarti, S. (2001). Invest. Ophthalmol. Vis. Sci. 42, 1750–1756. Maurice, D.M. (1957). J. Physiol. 136, 263–286. Meek, K.M., and Quantock, A.J. (2001). Prog. Ret. Eye Res. 20, 95–137. Meek, K.M., Quantock, A.J., Boote, C., Liu, C.Y., and Kao, W.W. (2003). Matrix Biol. 22, 467–475. Melki, S.A., and Azar, D.T. (2001). Surv. Ophthalmol. 46, 95–116. Newton, R.H., and Meek, K.M. (1998). Biophys. J. 75, 2508–2512.
Structure, Vol. 12, Feburary, 2004, 2004 Elsevier Science Ltd. All rights reserved.
Accessing Information on the Conformational Flexibility of Molecular Machines Acquiring information to describe the conformational flexibility of molecular machines is necessary to understand their mechanics but is technically difficult to achieve. In this issue of Structure, Chiu and colleagues effectively tackle this problem using 3D cryo-electron microscopy with impressive results.
The Problem At the heart of most biological processes, a molecular machine performs a given function or range of functions, generally by orchestrating a range of interactions with different cofactors. Molecular machines, the orchestra conductors, need to be physically flexible to accommodate all the necessary interactions and so must exhibit a certain degree of polymorphism. Obviously, a detailed
DOI 10.1016/j.str.2004.01.017
structural analysis of this polymorphism would shed much-needed light on the molecular mechanism that facilitates execution of a particular function. But how are these flexible intermediate structures to be analyzed and studied? More precisely, what experimental techniques can capture an image of a megadalton flexible complex and reveal its three-dimensional structure? The answer: certainly not many. In fact, three-dimensional cryo-electron microscopy (3D-cryoEM) is currently the best technique available to access this information experimentally since it does not require specimens to be arranged in a periodic manner, which would be difficult to achieve with mixtures of conformations, and it can deal with very large complexes. However, common image-processing approaches in the field do not work well if the sample is polymorphic. Obviously, for 3D-cryoEM to deliver, the analytical methods have to be improved, which is precisely the topic of Chiu and coworkers’ contribution published in this issue (Brink et al., 2004). The Prior Context in the Field of 3D-cryoEM A number of approaches to add the “flexibility” dimension to 3D-cryoEM studies have been considered. One approach directly works at the level of sample prepara-