To bead or not to bead? Lens-specific intermediate filaments revisited

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

Two type XII-like collagens localize to the surface of banded collagen fibrils.

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils.

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.

Observations on the Collagen and Proteinpolysaccharide Complex of Rabbit Corneal Stroma

The form and interrelationship of the collagen fibrils and proteinpolysaccharide complex of rabbit corneal stroma were studied by electron microscopy. The intact tissue was examined as Araldite sections stained with alkaline lead citrate and uranyl acetate, and the mechanically disintegrated cornea after positive or negative staining with phosphotungstic acid or after treatment with 0.5% bismuth nitrate in 0.1 M nitric acid. The corneal collagen fibrils vary in cross-sectional area from 4.6 to 9.6 x 104 sq. Å and do not exhibit a regular hexagonal distribution. Like tendon fibrils they consist of longitudinal filaments, but their appearance suggests that they lack some of the interfilament cross-links present in tendon. In sections of intact cornea and in negatively stained disintegrated cornea, filaments which are considered to be the protein cores of proteinpolysaccharide macromolecules are evident. They are about 40 Å wide and 2000 Å long. They appear to run an angular course, orthogonal to the collagen fibrils, and to be tangentially attached to several fibrils in the region of the a band. After treatment with bismuth nitrate disintegrated cornea contains coarsely beaded filaments. The filaments are about 2000 Å long and the beads about 70 Å in diameter. It is considered that these are again proteinpolysaccharide macromolecules and that each bead represents one or more polysaccharide chains in coiled configuration.

THE AMEBA-TO-FLAGELLATE TRANSFORMATION IN TETRAMITUS ROSTRATUS

Tetramitus exhibits independent ameboid and flagellate stages of remarkable morphological dichotomy. Transformation of the ameba involves the formation of four kinetosomes and their flagella. The arrangement of these kinetosomes and associated whorls of microtubules extending under the pellicle establishes the asymmetric flagellate form. While no recognizable kinetosomal precursors have been seen in amebae, and there is no suggestion of self-replication in dividing flagellates, developmental stages of kinetosomes have been identified. These are occasionally seen in association with the nucleus or with dense bodies which lie either inside of or close to the proximal end of the prokinetosome. Outgrowth of flagella involves formation of an axoneme and a membrane. From the distal tip of the kinetosome microtubules grow into a short bud, which soon forms an expanded balloon containing a reticulum of finely beaded filaments. The free ends of the microtubules appear unraveled; they are seen first as single elements, then as doublets, and finally are arranged into a cylinder. Growth in length is accompanied by a reduction in the diameter of the balloon. The concept that the formation of the kinetic apparatus might involve a nuclear contribution, followed by a spontaneous assembly of microtubules, is suggested.

Electron-Microscopic Studies on a Double-Stranded Beaded Filament of Embryonic Collagen

Beaded filaments consisting of paired 40-Å strands in parallel coupled by 150-Å beads at periodic spacings of 640 Å have been isolated from embryonic pig dermis. These filaments are found in association with embryonic collagenous fibrils and exclusively at sites of active fibrillogenesis of collagen. They have been identified within sectioned native fibrils as well as in interfibrillar regions. Furthermore, they have been recovered from fibrils denatured by exposure to alkalinity or to heat. In those instances, the filaments are packed in parallel array and the component beads are registered to produce a banding pattern with a major 640 Å repeat. The strands of the complex are composed of non-rigid fibrous protein as deduced from their fixation with aldehyde fixatives and from their degradation by urea. The beads are also proteinaceous, as indicated by their susceptibility to tryptic digestion. Moreover, these beads contain lipid, since they stain with lipid stains and dissolve in lipid solvents. Additional evidence suggests the inclusion of polysaccharide in the beads: periodic acid destroys these structures, and in native fibrils silver staining for carbohydrate occurs at loci of registered beads. The structural integrity of the conjugated protein bead does not depend upon the lipid or polysaccharide components, since lipases or β-amylase do not digest the structures. The filament proper is collagenase-resistant, but trypsin-sensitive. The latter enzyme bisects the beaded filament, forming single-stranded filaments with alternating 640-Å and 200-Å segments between beads. The double-stranded filaments are soluble in acid solutions below pH 4 but are stable at higher pH's through 9-10. The beaded filament fits into the scheme of extracellular collagen fibrillogenesis as the primary stable fibril or protofibril of collagen. It is presented as a doublet of procollagen fibrils which serves as a template for the polymerization of tropocollagen into additional beaded filaments. Aligned in parallel array and in register as they are assembled, the aggregate of beaded filaments is built into an immature fibril. By the establishment of interfilamentous cross-links, the individuality of the beaded filament is lost within the progressively maturing fibril.