Sieve structure of slit diaphragms of podocytes and pore cells of gastropod molluscs

Summary The ultrastructure of the slit diaphragms between the pedicels of the podocytes of the prosobranch Viviparus viviparus and between the cytoplasmic tongues of the haemocyanin producing pore cells of the pulmonate Lymnaea stagnalis was investigated. In both cell types 2 diaphragms are present in the slits. They form a 3-dimensional sieve structure with holes of respectively 90 × 110 Å (podocyte) and 200 × 220 Å (pore cell). Injection experiments showed that the size of the holes of the pore cell sieve matches that of particles which can be ingested by this cell type. The substructure of the sieves of the molluscs is compared to that of the 2-dimensional sieve of the podocytes of the mouse and the rat.The authors thank Mrs. J.E. Vlugt-van Dalen for technical assistance and Mr. G.W.H. van der Berg for drawing the diagrams. Thanks are furthermore due to Miss B.E.C. Plesch for correcting the English text     

Annulate lamellae in hindgut epithelial cells ofIsopoda

Summary Hindgut epithelial cells of the aquatic isopod,Asellus communis, and the terrestrial isopod,Armadillidium vulgare, possess annulate lamellae. The organelle is present in non-dividing cells of intermolt adult organisms. The lamellae exhibit dense pore areas traversed by diaphragms, and the lamellae are associated with elements of endoplasmic reticulum.This research was supported in part by a grant to E. R. Witkus from the Irene Heinz and John La Porte Given Foundation.     

Skeletal Ultrastructure in Some Articulate Cyclostome Bryozoans

The ultrastructure of the calcareous skeleton is described in 11 species of articulate cyclostome bryozoans with elastic joints. Ten species have interior walls comprising semi-nacreous and pseudofoliated fabrics without a precursory granular layer. Exterior walls consist of outer, finely granular and planar spherulitic layers, succeeded by semi-nacreous and pseudofoliated fabrics like those of interior walls. Outer fabrics are calcified as longitudinal strips, each corresponding to a planar sphcrulitic unit. Articulation surfaces comprise ring diaphragms of very fine granular fabric with concentric laminations. The semi-nacre of walls adjacent to ring diaphragms contains minute holes. Crisulipora occidentalis is unique in having interior walls of transverse fibres succeeded by pseudofoliated fabric, articulation surfaces festooned with deep pits but lacking well-differentiated ring diaphragms, and pseudopores containing sieve-like closure plates. The ultrastructure of most articulates resembles tubuliporine cyclostomes with dominantly semi-nacreous walls, although the lack of precursory granular fabric in the interior walls and the presence of subcircular tablets of semi-nacre (without six-fold sectoring) may be peculiar to articulates. In contrast, Crisulipora is more similar to other tubuliporines with transverse fibres. evidence which, together with other skeletal characters, suggests that Crisulipora evolved jointing independently of the rest of the articulate cyclostomes.     

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC)

1 Abbreviations: NPC, nuclear pore complex; GlcNac, N-acetylglucosamine; WGA, wheat germ agglutinin

. Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the inteaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the ‘gatekeepers’ that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests – perhaps.     

An Essential Role of the Universal Polarity Protein,aPKCλ, on the Maintenance of Podocyte Slit Diaphragms

Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin–podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3–atypical protein kinase C (aPKC)–PAR6β cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKCλ in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC–PAR3 complex associates with the nephrin–podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.     

Multiple origins of a unique pollen feature: stellate pore ornamentation in Amaranthaceae

Stellate pore ornamentation is an unusual feature of angiosperm pollen, so far it is known in only ten genera of Amaranthaceae. The pollen grains of these plants have apertures with large hook‐shaped ektexinous bodies that are stellately arranged. Previous studies interpreted this character complex as a synapomorphy in consideration of its strong specialization. By reconstructing the evolution of stellate pore ornamentation based on phylogenetic trees of Amaranthaceae obtained by parsimony, likelihood, and Bayesian analysis of chloroplast trnK/matK DNA sequences, clear evidence is provided for several independent origins and reversals to less specialized aperture types. In addition to the gomphrenoid genus Pseudoplantago, stellate pore ornamentation evolved several times among achyranthoid genera, which have an African distribution. The most derived apertures, with 5 – 6 large protruding hooks, appear independently in Centemopsis on the one hand, and in Psilotrichum sericeum on the other. In an effort to break down the complex character syndrome of stellate pore ornamentation, we delimited a set of six pollen morphological characters that could be independently traced on the phylogeny. It appears that stellate pore ornamentation was independently derived from apertures with equally spread ektexinous bodies that became hook‐shaped, reduced in number, and symmetrically arranged. Likewise the symmetrically arranged, rectangular ektexinous bodies in Marcelliopsis represent an independent specialization. In view of this pattern of morphological changes, functional significance in the context of specialized insect pollination syndromes and positive selection for stellate pore ornamentation is hypothesized. Stellate pore ornamentation provides an example of a specialized pollen character complex with adaptive significance, and underlines the need for a dense taxon sampling for analyses of character evolution.     

Ultrastructure of Stylodinium Iittorale (Dinophyceae) with special reference to the stalk and apical stalk complex

The ultrastructure of Stylodinium littorale Horiguchi et Chihara, a marine, sand-dwelling coccoid dinoflagel-late, was investigated with special emphasis on its stalk and the apical stalk complex. The dinoflagellate alternates between non-motile and motile cells in its life cycle. The non-motile cell possesses a long and distinct stalk. The stalk, consisting of a main cylindrical part and a holdfast, is firmly attached to a thecal plate (the apical pore plate). A part of its proximal portion is hollow and V-shaped in section. The V-shaped hollow space is underlain by a projection from the apical pore plate. An apical stalk complex is present in the motile cells and consists of a large apical pore plate and mucilaginous material. The apical pore plate is depressed into the cell, but has a narrow central tubular projection. The mucilaginous stalk-building material is stored between this plate and the outer plate membrane. The tubular projection of the apical pore plate corresponds to the apical pore of other dinoflagellates and its lumen is filled with electron-dense material. The structure of the apical stalk complex is compared with the homologous structure in Bysmatrum arenicola, the only other example of an apical stalk complex that has been investigated. A general ultrastructural survey revealed that S. littorale possesses a typical dinoflagellate cellular structure.     

ONTOGENY OF FOLIAR DIAPHRAGMS IN TYPHA LATIFOLIA

Morphogenetic pulsations in the intercalary meristem of the leaf of Typha latifolia (Typhaceae) produce regular alternating sequences of vascular and stellate-celled diaphragms separated at first by rib-meristem derivatives. The collapse of these derivatives in the region of elongation in and above the intercalary meristem, and the separation of the diaphragms from each other, produce a mature compartmentalized leaf, the compartments bridged by porous diaphragms but separated from each other by rigid vascularized partitions.     

DEVELOPMENT OF CHAMBERED PITH IN STEMS OF PHYTOLACCA AMERICANA L. (PHYTOLACCACEAE)

An integrated microscopic (light and electron microscopy) and macroscopic investigation of chambered pith development was made of Phytolacca americana L. Terminal internodes have a solid pith cylinder in contrast to the alternating diaphragms and chambers occurring in subjacent pith. Macroscopically, chambers and diaphragms of any one internode are of equal size. Microscopically, diaphragms vary in height within an internode (from 1–6 cells high). Nevertheless, all diaphragms become thicker circumferentially (5–12 cells high) and connect with long files of intact peripheral pith cells. Diaphragm cells have a large centrally positioned vacuole with a thin, parietal layer of cytoplasm; nuclei, mitochondria, endoplasmic reticulum, and unidentified organelles differentiate in the cytoplasm of diaphragm cells. Although schizogenous activity has most often been implicated as the mechanism by which chambered pith develops in vegetative organs of angiosperms, the results of this study show that cavities in pokeweed result from both schizogenous and lysigenous mechanisms. Schizogeny is suggested by the fact that central pith cells of terminal internodes are longer and thinner walled than peripheral pith cells arranged in vertical files, thus indicating elongation of cells as a possible result of internode elongation. The precise developmental pattern and arrangement of chambers and diaphragms also suggest schizogenous processes. Lysigenous or enzymatic activity is indicated by the fact that cavities are bounded by broken cells, and wall fragments and organelles are often found within enlarging cavities. Chamber formation occurs continuously acropetally and centrifugally in the central pith. A comparison of diaphragms is made with Liriodendron tulipifera and Juglans nigra in an attempt to resolve differences in structure and terminology regarding the differentiation of chambered and diaphragmed pith.     

DEVELOPMENT OF FOLIAR DIAPHRAGMS IN SPARGANIUM EURYCARPUM

Three types of diaphragms are produced in regular sequence by the basal intercalary meristem in the leaf of Sparganium eurycarpum Engelm. (Sparganiaceae). They bridge compartments formed by the collapse and disintegration of rib meristem derivatives. The adaptive nature of diaphragms, intercalary meristems, and linear photosynthetic organs is considered for emergent aquatic plants.     

Calculating Transition Energy Barriers and Characterizing Activation States for Steps of Fusion

We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.     

ADAPTIVE LEAF ARCHITECTURE IN EMERGENT AND FLOATING SPARGANIUM

The robust emergent leaves of Sparganium eurycarpum and S. americanum are supported by corner fiber masses and large bundle sheaths, but the thin floating leaves of S. fluctuans and S. minimum have only moderate bundle sheaths. In emergent types heavily photosynthetic diaphragms bearing vascular bundles are separated from each other in the leaf compartments by three lightly photosynthetic diaphragms without bundles, but in floating types only every other heavily photosynthetic diaphragm has a bundle. Palisade chlorenchyma occurs only at aerial surfaces—abaxial and adaxial in emergent leaves, but only adaxial in floating leaves. Extra photosynthetic areas are provided in emergent leaves by concentrations of chlorenchyma in limited areas on interior partitioning walls, while the remainder of the walls is translucent. Since only 25 % of the diaphragms are heavily photosynthetic, and the others essentially transparent because of their diffusely distributed chloroplasts and large intercellular spaces, a sieve effect exists which allows even the interior parts of thick emergent leaves to photosynthesize.     

Formation of intercellular gas space in the diaphragm during the development of aerenchyma in the leaf petiole of Sagittaria trifolia

The development of aerenchyma in the petiole of Sagittaria trifolia L. was studied by means of light-microscopy, scanning electron microscope, transmission electron microscope and immunofluorescence, focusing on the formation of intercellular spaces in diaphragms and its relationship with the organization of cortical microtubule arrays. A complex and organized honeycomb-like schizogenous aerenchyma formed by cylinders and vascular diaphragms was observed in the petiole of S. trifolia at different developmental stages. Cell division was the primary factor contributing to the increased volume of air spaces at early stages, while cell enlargement became the primary factor at later stages. The cortical microtubules localize at the sites where intercellular spaces and the secondary cell walls will be formed or deposited during the formation of intercellular spaces by the separation of diaphragm cells. Cortical microtubules were observed at the boundary of diaphragm cells and the fringes of intercellular spaces at later developmental stages where cell expansion occurs rapidly. These observations support the hypothesis that reorganization of cortical microtubule arrays might be related to the formation of air spaces in diaphragms and are involved in the deposition of secondary cell walls.     

Zooidal anatomy in Ordovician and Carboniferous trepostome bryozoans

Fossil bryozoans sometimes contain fossilised brown bodies which remain after polypide degeneration. Position and shape of brown bodies as well as different skeletal diaphragms within living chambers allow outlining of autozooid anatomy in Palaeozoic trepostome bryozoans. In some trepostome bryozoans short autozooids were restricted by complete basal diaphragms to distal parts of autozooecia. In species with alternating hemiphragms, as in the specimen ofHemiphragma sp. described here, long autozooids occupied the entire autozooecial chambers. Their short polypides were positioned in distal parts of the autozooid. Both anatomic types correspond to the progressive polypide cycle afterBoardman. For species with ring septa, non-alternating hemiphragms (studied in an example ofNipponostenopora karatauensis) as well as without any diaphragms the stationary type of polypide cycle seems also to be possible. In that case, the polypides should be as long as the cystid.      

Immunological localization of the major architectural protein associated with the nuclear envelope of the Xenopus laevis oocyte

The nuclear envelope (NE) of Xenopus laevis contains a major architectural protein which is resistant to extraction in high salt buffer and non-ionic detergent and is characterized by a polypeptide molecular weight (MW) of 68000. Two different antisera which showed specific binding of antibodies (IgG) to this polypeptide, as demonstrated by ‘immunoblotting’ techniques, were used for immunolocalization at the electron microscopic level. Whereas antibodies of serum I reacted only with the nuclear lamina structure, antibodies of serum II, which were raised against undenatured karyoskeletal protein from oocytes, showed additional strong reaction in nuclear pore complexes. This first positive localization of a polypeptide in the nuclear pore complex suggests that MW 68000 polypeptide contributes as a major karyoskeletal component to the structure of both the lamina and the pore complex.     

Anatomy and ultrastructure of the excretory system of a heart-bearing and a heart-less sacoglossan gastropod (Opisthobranchia, Sacoglossa)

The microanatomy and ultrastructure of the excretory system of the Sacoglossa have been investigated from two species by means of semithin serial sections, reconstructions and transmission electron microscopy. Whereas Bosellia mimetica shows a functional metanephridial system consisting of a heart with ventricle and auricle in a pericardium and a single kidney, Alderia modesta lacks heart and pericardium, possessing only several haemocoelic sinuses and a very long kidney. In B. mimetica podocytes as the site of ultrafiltration could be detected in the pericardial epithelium lining the auricular wall. The flat epithelium of the kidney with extensive basal infoldings and a dense microvillous border towards the luminal surface serves to modify the ultrafiltrate. In A. modesta podocytes are absent. Solitary rhogocytes (pore cells), the fine structure of which strongly resembles podocytes (meandering slits with diaphragms covered by extracellular matrix), occur in B. mimetica and A. modesta, representing additional loci of ultrafiltration. The presence of podocytes situated in the epicardial wall of the auricle is regarded as plesiomorphic for the Mollusca and confirmed for the Sacoglossa in this study, contradicting earlier assumptions of the loss of the primary site of ultrafiltration in the ancestors of the Opisthobranchia. In contrast to the likewise heart-less Rhodopidae with a pseudoprotonephridial ultrafiltration system, A. modesta shows no further modifications of the excretory system. Accepted: 7 May 2001     

Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition

SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.