Renal architecture of the dogfish Scyliorhinus caniculus (Chondrichthyes,Elasmobranchii)

Summary The excretory portion of the opisthonephric kidney of Scyliorhinus caniculus displays a mesial zone that is supplied with venous blood by the renal portal system and with arterial blood from the efferent arterioles of the glomeruli, and a zone of lateral bundles that is irrigated with arterial blood via arterioles in parallel to the afferent arterioles of the glomeruli. Each single nephron performs two large convolutions in the mesial tissue and two hairpin loops in the bundle. The nephron is differentiated into renal corpuscle (located between the two zones), neck segment (in the bundle), proximal segment I (beginning in the bundle, major convolution between the zones), proximal segment II (exclusively in the mesial zone), intermediate segment (beginning in the mesial tissue and ending in the bundle), distal segment (exclusively in the bundle) and collecting tubule (beginning in the bundle, with a large convolution in the mesial tissue and ending in the bundle) that joins the collecting duct-ureter system. In the bundles proximal and distal nephron segments, the end of the renal tubule and a central bundle vessel are arranged together and form a complex countercurrent system that is enclosed in a sheath of connective tissue. The bundles provide the structural basis for the creation of an environment with low urea concentration around the final portion of the renal tubules, which is consistent with previous experimental evidence of a significantly lower urea content of the bundles as compared with the blood and the mesial tissue in another marine elasmobranch, Raja erinacea. This condition is thought to lead to passive reabsorption of urea from the fluid of the end of the renal tubule. Separation of individual nephrons in the bundle zone appears to be correlated with the peculiar secondary structure that results from the folding of the bundles and may be in addition a requirement in conjunction with intermittent function of the glomeruli. The zonation of the renal tissue with formation of bundles with counter-current systems is characteristically found in marine Elasmobranchs and is considered to be the morphological correlate to the physiological ability of the marine Elasmobranchii to use urea for osmoregulation.     

Vascular Bundle Distribution Within Broad Bean Seed Coat and the Structure of Unloading Region

Cytological observations were made on developing seed coat of broad bean (Vicia faba L.) by use of light and electron microscopy. Attention was focused on vascular distribution. The seeds were attached by the funiculus to tile large vascular bundles of pericarp of broad bean. The vascular bundle passed through hilum and two layers of palisade, entered the pa- renchyma of seed coat, then diverged in to two routes. One was a complete vascular bundle composed of both'phloem and xylem elements, it stretched down through seed raphe, then upward and terminated near the radical. The other was a two-recurrent-vascular-bundle with only phloem constitutents, they extended forward detoured the micropyle and extended downward, but did not join with the upward complete vascular bundle. The recurrent vascular bundles branched out many small short branches. The obvious difference between phloem of recurrent vascular bundle and of complete vascular bundle was that the companion ceils of the former did not normally modify to transfer ceils, but connected to the adjoining parenchyma cells through abundant plasmodesmata. It is deduced from the structural analysis that the symplastic route may play an important role, particularly in the region of recurrent vascular bundle, in the course of importing assimilates unloading in seed coat and transporting to the embryo.     

Transiently crosslinked F-actin bundles

F-actin bundles are prominent cytoskeletal structures in eukaryotes. They provide mechanical stability in stereocilia, microvilli, filopodia, stress fibers and the sperm acrosome. Bundles are typically stabilized by a wide range of specific crosslinking proteins, most of which exhibit off-rates on the order of 1s⁻¹. Yet F-actin bundles exhibit structural and mechanical integrity on time scales that are orders of magnitude longer. By applying large deformations to reconstituted F-actin bundles using optical tweezers, we provide direct evidence of their differential mechanical response in vitro: bundles exhibit fully reversible, elastic response on short time scales and irreversible, elasto-plastic response on time scales that are long compared to the characteristic crosslink dissociation time. Our measurements show a broad range of characteristic relaxation times for reconstituted F-actin bundles. This can be reconciled by considering that bundle relaxation behavior is also modulated by the number of filaments, crosslinking type and occupation number as well as the consideration of defects due to filament ends.     

The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor

Macromolecules and cells exposed to blood flow in the circulatory tree experience hydrodynamic forces that affect their structure and function. After introducing the general theory of the effects of shear forces on protein conformation, selected examples are presented in this review for biological macromolecules sensitive to shear stress. In particular, the biochemical effects of shear stress in controlling the von Willebrand Factor (VWF) conformation are extensively described. This protein, together with blood platelets, is the main actor of the early steps of primary haemostasis. Under the effect of shear forces > 30 dyn/cm², VWF unfolding occurs and the protein exhibits an extended chain conformation oriented in the general direction of the shear stress field. The stretched VWF conformation favors also a process of self aggregation, responsible for the formation of a spider web network, particularly efficient in the trapping process of flowing platelets. Thus, the effect of shear stress on conformational changes in VWF shows a close structure-function relationship in VWF for platelet adhesion and thrombus formation in arterial circulation, where high shear stress is present. The investigation of biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular interaction mechanisms involved in arterial thrombosis.     

Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin

Talin is an adaptor protein that couples integrins to F-actin. Structural studies show that the N-terminal talin head contains an atypical FERM domain, whereas the N- and C-terminal parts of the talin rod include a series of α-helical bundles. However, determining the structure of the central part of the rod has proved problematic. Residues 1359–1659 are homologous to the MESDc1 gene product, and we therefore expressed this region of talin in Escherichia coli. The crystal structure shows a unique fold comprised of a 5- and 4-helix bundle. The 5-helix bundle is composed of nonsequential helices due to insertion of the 4-helix bundle into the loop at the C terminus of helix α3. The linker connecting the bundles forms a two-stranded anti-parallel β-sheet likely limiting the relative movement of the two bundles. Because the 5-helix bundle contains the N and C termini of this module, we propose that it is linked by short loops to adjacent bundles, whereas the 4-helix bundle protrudes from the rod. This suggests the 4-helix bundle has a unique role, and its pI (7.8) is higher than other rod domains. Both helical bundles contain vinculin-binding sites but that in the isolated 5-helix bundle is cryptic, whereas that in the isolated 4-helix bundle is constitutively active. In contrast, both bundles are required for actin binding. Finally, we show that the MESDc1 protein, which is predicted to have a similar fold, is a novel actin-binding protein.     

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)₃ that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of ∼21 pN and ∼63 nm, respectively. Stretching of (A1A2A3)₃ yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.     

Tissue mechanical behavior of tendinous fibers under statistical and dynamic requirements

With a device for dynamical processes tension tests were performed on bundles of collagen fibres of human and bovin origin. Part of the studies were performed under statical condition with an universal materials testing machine, equipped with a closed loop feed back control system. If the force is to be kept constant subsequently to a strain process on a collagen fiber bundle (isotonic condition), the fiber length must increase (creep phenomenon, retardation). The force decreases under constant length after a preceding strain process (relaxation). In analogy to the statical relaxation, statical isorheological line, and statical force recovery curve the dynamical (cyclic) relaxation, dynamical (cyclic) isorheological curve, and dynamical (cyclic) force recovery curve are described. The mechanical-rheological properties collagen fiber bundles are discussed in relation to functional anatomy.     

Comparative morphology of the carpel in the Liliaceae: Glorioseae

The pistils of the Glorioseae (Gloriosa, Littonia, Sandersonia) are generally tricarpellate and alike. Virtually all have closed sutures at flowering; they have many ovules, some of which are barely bitegmic, with inner integuments often nearly fused with nucellar remnants; and there is usually but one compound septal bundle in the inner edge of a septum. In two species of Littonia , the compound septal bundle divided to form two simple septal bundles; but in many other plants it remained undivided, and in some it died out, still undivided, below the locular apex. Most of the placental and septal bundles are vascularized in large part by three alternate (compound septal) bundles at the base of the locules and sometimes by branches from the lateral bundles. Three large (compound) placental bundles are formed just below the lowermost ovular insertion, and each then divides in two to furnish ovular branches along their ascent. Occasional auxiliary placental bundles lie between the septal bundle and the placental bundles in the septum (Gloriosa, Sandersonia).     

Two adaptation processes in auditory hair cells together can provide an active amplifier

The hair cells of the vertebrate inner ear convert mechanical stimuli to electrical signals. Two adaptation mechanisms are known to modify the ionic current flowing through the transduction channels of the hair bundles: a rapid process involves Ca(2+) ions binding to the channels; and a slower adaptation is associated with the movement of myosin motors. We present a mathematical model of the hair cell which demonstrates that the combination of these two mechanisms can produce "self-tuned critical oscillations", i.e., maintain the hair bundle at the threshold of an oscillatory instability. The characteristic frequency depends on the geometry of the bundle and on the Ca(2+) dynamics, but is independent of channel kinetics. Poised on the verge of vibrating, the hair bundle acts as an active amplifier. However, if the hair cell is sufficiently perturbed, other dynamical regimes can occur. These include slow relaxation oscillations which resemble the hair bundle motion observed in some experimental preparations.     

Structural hierarchy of mechanical extensibility in human von Willebrand factor multimers

The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C_1–6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A₂ domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.     

A Simple Clear Technique in Observing Vascular Development of Grape Ovary

The vascular system of the grapevine (Vitis vinifera L.) flower is a channel for transporting water and nutrients to the ovary. It plays an important role in the development of the ovary and fertilization through pollination. However, the vascular bundles in the flower are so tiny that they are difficult to sample and observe by traditional slicing techniques. In this study, ‘Summer Black’ grape flowers were selected as the test materials, and the tissue samples were treated by the optical clearing technique. After simple compaction, the structure and development of the vasculature were observed by common microscopy, fluorescence microscopy and laser confocal microscopy. The results showed that the transparency effects of 3% NaOH and a saturated trichloroacetaldehyde composite agreed well with the observations of the vascular structure and the developmental process of the flower in different periods. Moreover, the samples after optical clearing could be reconstructed in 3D, which helped us know more about its development and function. According to these observations, the vasculature of the ‘Summer Black’ flower can be divided into ovule vascular bundles, peripheral vascular bundles and central vascular bundles. The peripheral vascular bundles were composed of the first-order vascular bundles and the inferior vascular bundles which branched from the superior vascular bundles. These bundles branched in different directions with no discernible pattern. The two different branching methods were as follows. First, the inferior vascular bundle was directly connected to a superior vascular bundle. Secondly, some of the superior vascular bundles bent in different ways, forming the inferior vascular bundle connecting the superior vascular bundles by a metamorphosed vessel with a triangular shape. In a comparison of the developmental changes in various periods, the growth of vascular bundles at each period was directly proportional to the growth of the flower. Laser confocal scanning was used to explore the three-dimensional morphology of the peripheral vascular bundle and showed that the peripheral vascular bundle of grapes was not completely parallel to the flower’s epidermal cells. As a result, the optical clearing technique was convenient and authentic compared with the traditional slicing operation for tiny flower organs. With these advantages according to the observations, this study provides a feasible technique and useful information for the study of vascular bundle development in grape flower organs.     

On the eye of the Goldeye Hiodon alosoides (Teleostei: Hiodontidae)

In the eye of the Goldeye the photoreceptors are arranged in bundles and the pigment epithelium contains a massive reflector or tapetum lucidum. Photoreceptor bundles are arranged in parallel rows, the bundles alternating in position from row to row. Each bundle contains about 60 photoreceptors, of which 30 or so are cones. Rod outer segments lie in the scleral half of the outer retinal region of the light-adapted eye. Processes of the pigment epithelium cells extend vitread almost to the external limiting membrane; they envelop the bundles of rods and cones, and a ring of four processes surrounds each bundle. A process contains two kinds of reflecting crystals (composed of uric acid). A large part of the epithelium cell is packed with small disc-shaped crystals (crystallites) enclosed in thin membranes; the tip of the process, in the region of the photoreceptor bundle, contains orderly arrays of small rod-shaped crystals (rodlets). It is suggested that the crystallites form a diffuse reflector backscattering light into the rods; and that the rodlets reflect light regularly from their surfaces into the photoreceptor bundles. In the light-adapted state, rods are enveloped by pigment and crystallites. The organization is compared with that of other fishes that have photoreceptors in bundles (grouped retinae) and tapeta lucida.     

Comparative morphology of the carpel in the Liliaceae: Baeometra, Burchardia and Walleria

The pistils in Baeometra, Burchardia and Walleria ate tricarpellate, and their ovules are mostly bitegmic. Baeometra has free styles and deep septal invaginations between the carpels. Its pistil is innervated by three dorsal bundles, three compound septal bundles (each of which may divide into two simple septal bundles above), six placental bundles, and six adjoining auxiliary placental bundles. The pistil of Burchardia resembles that of Baeometra , except that there are six simple septal bundles throughout and no auxiliary placental bundles. In Walleria the wings of adjoining carpels are completely fused (except for rare septal glands); there is a single compound style; additional vascular tissue is present in the central axis of the pistil up to the lowermost ovules; the carpels are fused with the floral cup above the base of the locules; and raphide idioblasts are present. Walleria has six "ventral" bundles, each of which appears to be the fusion product of a placental bundle with a simple septal bundle. Tribal affinities of these genera are discussed.     

Morphology ofCoptis japonica and its meaning in phylogeny

In the genusCoptis, some interesting features are found which are considered important phylogenetically. The median bundles of petiole and petiolule, and the midrib of lamina are double. They seem to represent a transitional situation between a dichotomy and a single median bundle found in usual angiospermous venation. The double bundle is either derived from 2 independent leaf trace bundles or formed by dichotomy of a leaf trace bundle, and it does not seem so important whether the number of trace bundles is even or odd. The nodal structure is trilacunar or pentalacunar with 3, 4, 6 or 8 trace bundles. The upper part of the carpel does not produce ovules and is open from the initiation of the carpel. It is suggested that the carpel becomes open secondarily concomitant with the reduction of ovules. This shows that the closure of the carpel is not perfectly established.     

Sound-induced motions of individual cochlear hair bundles

We present motions of individual freestanding hair bundles in an isolated cochlea in response to tonal sound stimulation. Motions were measured from images taken by strobing a light source at the tone frequency. The tips and bases of hair bundles moved a comparable amount, but with a phase difference that increased by 180 degrees with frequency, indicating that distributed fluid properties drove hair bundle motion. Hair bundle rotation increased with frequency to a constant value, and underwent >90 degrees of phase change. The frequency at which the phase of rotation relative to deflection of the bundle base was 60 degrees was comparable to the expected best frequency of each hair cell, and varied inversely with the square of bundle height. The sharpness of tuning of individual hair bundles was comparable to that of hair cell receptor potentials at high sound levels. These results indicate that frequency selectivity at high sound levels in this cochlea is purely mechanical, determined by the interaction of hair bundles with the surrounding fluid. The sharper tuning of receptor potentials at lower sound levels is consistent with the presence of a negative damping, but not a negative stiffness, as an active amplifier in hair bundles.     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Nonlinear mechanical responses of mouse cochlear hair bundles.

The stiffness of sensory hair bundles of both inner (IHC) and outer (OHC) hair cells was measured with calibrated silica fibres in mouse cochlear cultures to test the hypothesis that the mechanical properties of the hair bundle reflect processes underlying mechanotransduction. For OHCs, the displacement of the hair bundle relaxed with time constants of 6 ms for displacements which open transducer channels and 4 ms for displacements which close the channels. The corresponding values of the time constants for IHCs were 10 ms and 8 ms, respectively. A displacement-dependent change in the stiffness of the hair bundle was not observed when the bundle was displaced orthogonally to the direction of excitation. The stiffness of the hair bundle as a function of nanometre displacements from the resting position was remarkably nonlinear. The stiffness declined to a minimum from the resting stiffness by about 12% for OHCs and 20% for IHCs when the hair bundle was displaced by about 20 nm in the excitatory direction, and it increased by a similar amount when the bundle was displaced by 20 nm in the inhibitory direction. The displacement at which the stiffness reached a minimum was within the most sensitive region of the hair-cell transducer function (receptor potential as a function of hair-bundle displacement), and the displacement at which the stiffness reached a maximum was at the point of saturation of the transducer function in the inhibitory direction. The nonlinear displacement-dependent compliance change is reversibly abolished, and the time constant of relaxation of the bundle for excitatory displacements is reversibly reduced, when mechanotransduction is blocked by the addition of either neomycin sulphate or cobalt chloride to the solution bathing the hair cells. The displacement-dependent compliance change was not apparently reduced when the receptor potential was attenuated through the substitution of sodium in the bathing solution with a less permeant cation, tetraethylammonium. These findings suggest that the nonlinear mechanical properties of the hair bundle are associated with aspects of the hair-cell mechanotransducer process. The mechanical properties of the hair bundle are discussed in relation to the 'gating-spring' hypothesis of hair-cell transduction.     

F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.     

PROTOXYLEM MATURATION IN THE SEEDLING LEAF OF WHEAT

Protoxylem (PX) maturation was followed in vascular bundles of the first foliage leaf (L#1) of wheat seedlings, using clearings and sectioned materials to assess development changes. L#1 contained seven to 11 longitudinal bundles. The seven bundles with largest diameters contained PX vessels, but the number of bundles with PX varied with leaf length. Repeated vessel maturation maintained PX continuity as older vessels collapsed due to leaf extension. Thus, the number of PX vessels in each bundle was also a function of leaf length, and the PX content of a bundle was a function of the stage of leaf development and how soon during leaf development the bundle attained mature PX. Distance from the leaf tip to the start point for PX was greater for bundles later to mature. Thus, the pattern of distal start points in mature leaves reflected the progression of PX maturation from the midrib to bundles of lower rank. It is suggested that the involvement of more and more bundles in PX maturation as the leaf ages is a general occurrence in grass leaves, and that assessing how many bundles will have PX requires observations late in leaf development.     

Polarized actin bundles formed by human fascin-1: their sliding and disassembly on myosin II and myosin V in vitro

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.