The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule-based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal-most region of the axoneme, termed the transition zone, which is characterized by Y-shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.     

From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility

Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.     

Tumour necrosis factor blockade and the risk of osteoporosis: back to the future

Osteoporosis is a common clinical problem, especially in patients with rheumatoid arthritis (RA). A reduction in bone mineral density (BMD) of the axial and appendicular skeleton ranging from 7% to 15% has been reported in RA in studies employing a variety of densitometric techniques. Reports consistent with a beneficial effect of tumour necrosis factor blockade on BMD have begun to emerge in recent years, and in Arthritis Research and Therapy, a case control study reports that patients treated with infliximab for RA had preservation of BMD in the lumbar spine and femoral neck compared to those treated with methotrexate.     

Ultrastructure of Amoebophrya sp. and its changes during the course of infection

Amoebophrya is a syndinian parasite that kills harmful bloom forming algae. Previously uncharacterized ultrastructural aspects of infection and development were elucidated. The biflagellate dinospore has two mitochondria, electron-dense bodies, striated strips, trichocysts, and a nucleus with peripherally condensed chromatin. After finding an Akashiwo sanguinea host and adhering to its surface, the parasite penetrates the host surface, apparently using a microfilament based motility and electron-dense bodies within a microtubular basket in the process of parasitophorous vacuole membrane formation. After entering the host nucleus, possibly by a similar mechanism used to enter the host cell, the parasite cytosol expanded substantially prior to mitosis. From 12-36 hours mitochondria were inconspicuous but present. Chromatin condensation was variable. By 36 hours post-infection, parasites had multiple nuclei, a microtubule-supported cytopharynx, and were beginning to form a fully internal mastigocoel. By 48 hours, the characteristic "beehive" appearance was apparent with flagella projecting into a fully developed mastigocoel. The cytoplasm contained trichocysts, elongated mitochondria, and nuclei with peripherally condensed chromatin. Although Amoebophrya lacks an apical complex, its electron-dense bodies show functional similarities to apicomplexan rhoptries. Its lack of permanently condensed chromosomes, but compact dinospore chromatin, supports the idea that dinoflagellate permanently condensed chromosomes may be a remnant of a parasitic ancestor with a compact dispersal stage.     

鸡脊髓背角胶状质中calbindin-D28k阳性终末的超微结构及与substance P阳性终末之间的联系

目的 观察鸡脊髓背角胶状质中calbindin-D28k（CB）阳性终末的超微结构及其与含有substance P（SP）中央末梢之间的联系.方法 应用免疫电镜技术观察鸡脊髓背角胶状质中CB阳性终末的超微结构,并应用激光共聚焦显微镜观察鸡脊髓背角胶状质中CB和SP阳性突触小球中央末梢之间的关系.结果 电镜下观察：1）突触小球中含有心小泡的中央末梢呈CB免疫阳性;2）突触小球内或外的部分含小泡的树突呈CB免疫阳性;以及3）突触小球外的部分轴突呈CB免疫阳性.在突触结构内,CB免疫阳性反应物主要分布于突触后膜上.免疫荧光双标记法显示,SP阳性的含有心小泡的中央末梢呈CB阳性.结论 突触小球的中央末梢中CB与SP共存,提示CB可能通过其钙离子缓冲作用,参与脊髓的痛觉调制.     

Effects of the Menopause Transition on Body Fatness and Body Fat Distribution

Objective : The menopause transition increases cardiovascular and metabolic disease risk, partly because of the adverse effects of estrogen deficiency on the plasma lipid-lipoprotein profile and cardiovascular function. This increased cardiovascular and metabolic disease risk may also be partially mediated by increased body fat, increased intraabdominal adipose tissue accumulation, or both. The objective of this mini-review is to summarize studies that have investigated the relationships among the menopause transition, body fatness, and body fat distribution. Research Methods and Procedures : A review of cross-sectional and longitudinal studies on menopause that examined body fatness and body fat distribution. Results : Cross-sectional reports show that the menopause transition is related to modest increases in body mass index or total fatness, although not all studies found significant effects. Increased central adiposity appears to be related to menopause, independent of advancing age, but these results are methodology dependent. An independent effect of menopause on central body fatness was noted by the use of techniques such as DEXA or computed tomography, whereas studies using circumference measures showed discrepant results. Longitudinal studies showed that the menopause transition accelerated the increase in central adiposity, although no studies quantified changes in intra-abdominal fat by imaging techniques. Discussion : Thus, additional longitudinal studies using more accurate measures of adiposity are needed to critically examine the effects of the menopause transition on total and central body fatness. Collectively, previous studies suggest that menopause is related to modest increase in total fatness and accelerated accumulation of central body fat that exceeds changes normally attributed to the aging process. These changes may increase the risk for cardiovascular and metabolic disease in aging women.     

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-β CH₂ group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the β-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the β-phosphate with BH₃ or Se, or substituted at either the α-β or β-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m₂^7,2′-OGpp_SpG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m⁷Gpp_BH3pm⁷G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t_1/2 ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.     

Comparative histochemical observations on the contributions of the acrosomal vesicle and granule to the acrosomal cap in the spermatozoa of mammals

Summary By applying the periodic acid-Schiff technique to frozen sections of material fixed in weak Bouin's solution and extracted with hot pyridine, the contributions of the acrosomal vesicle and granule to the acrosomal cap have been observed in the maturing spermatozoa of the American opossum, guinea pig, rabbit, marmoset and chimpanzee. In the spermatids of the opossum, the acrosomal granule is not developed and the thin, homogeneous carbohydrate layer of the acrosomal cap is derived from the concentrated material of the acrosomal vacuole. In the guinea pig, both the acrosomal vesicle and granule make the greatest contributions to the carbohydrates of the acrosomal cap; these continue to maintain their identity in the head of mature sperm. In the rabbit, marmoset and chimpanzee, the carbohydrate material of acrosomal vesicle origin is less developed and constitutes a thin layer in the lateral portions of the acrosomal cap; the carbohydrate material in its anterior portions is mainly derived from the acrosomal granule which becomes flattened; there is also a slight thickening of the intermediate layer of the acrosomal cap in this region. The distinction between the carbohydrate contributions from the acrosomal vesicle and granule disappears in the fully formed acrosomal cap which shows a homogeneously PAS-positive intermediate layer. In the chimpanzee, the acrosomal vesicle makes a relatively very small contribution to the carbohydrate layer of the acrosomal cap which is mainly formed by spreading of material from the acrosomal granule.     

Structure Analysis of the Flagellar Cap–Filament Complex by Electron Cryomicroscopy and Single-Particle Image Analysis

The cap of the bacterial flagellum plays an essential role in the growth of the long helical filament by promoting the efficient self-assembly of flagellin transported to the distal end through the narrow central channel of the flagellum. The structure of the cap–filament complex was analyzed by electron cryomicroscopy and single-particle image analysis to understand how the cap stays attached while allowing the flagellin insertion between the cap and the filament end and also allowing the HAP proteins to pass through. In the images of the complex, the projection pattern of the helical subunit array in the filament portion occupied the major fraction but was variable depending on the azimuthal orientation of the filament; therefore the images showed a strong tendency to be misaligned. Various methods had to be newly developed to correctly align the images by overcoming this misalignment problem. The structure thus obtained clearly demonstrated the pentameric structure of the cap and how the cap operates. The new methods of analysis presented here would be generally applicable to cap structures of various filaments that play biologically important roles in cellular activities.     

A Trypanosoma brucei protein required for maintenance of the flagellum attachment zone and flagellar pocket ER domains

Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the 'FAZ ER'. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.     

Ultrastructure and cell size-dependent regulation of the adoral zone of membranelles in the heterotrich ciliate Stentor coeruleus

The number and length of oral membranelles were determined for both large and small Stentor from well-fed, growing cultures and nutrient-deprived cultures, respectively. Small cells possess both significantly fewer and shorter membranelles than do large cells. For both large and small cells, each membranelle is composed of three rows of basal bodies. The membranelles closest to the gullet have a third row that is only slightly shorter than the other two. The third row becomes rapidly shorter as membranelles become increasingly distant from the gullet. A short distance from the gullet, and for the remainder of the band, the third row is composed ofonly one to four basal bodies. The first two rows consist of approximately 35 basal bodies each in large cells and approximately 26 basal bodies each in small cells. This indicates that Stentor regulates the number of basal bodies per row, but not the number of rows, in response to changes in cell size. © 1992 Wiley-Liss, Inc.     

Toward Conceptual Cohesiveness: a Historical Analysis of the Theory and Utility of Ecological Boundaries and Transition Zones

Ecological transition zones are increasingly recognized as systems that play a critical role in controlling or modifying flows of organisms, materials, and energy across landscapes. Many concepts describing transitional areas have been proposed over the years, such as the prevalent and durable ecotone concept. Confusion among ecologists and land managers about transition zone concepts and the isolation of studies that use only one transition concept can hinder unified progress in understanding these key systems. Currently, a movement toward conceptual synthesis under the umbrella concept of ‘ecological boundary’ is underway. Here we examine the history and theoretical baggage of the ecotone, riparian zone, and several other concepts. Subsequently, we present a conceptual cluster analysis, which facilitates a better understanding of the similarities and differences between boundary and transition concepts. We emphasize the hierarchical nature of these concepts: higher-level synthetic concepts can be used in the development of theory, whereas lower-level concepts allow more specificity and the formulation of operational definitions. Finally, we look briefly at the utility and future use of boundary and transition zone concepts.     

气候变暖与荒漠-草原过渡带植物动态响应灰色分析--以美国新墨西哥州Sevilleta生态过渡带为例

在当前全球气候变暖的形势下,地表生态系统表现出不同的响应。干旱-半干旱地区的生态过渡带的生物反应更为敏感。美国新墨西哥州中部的荒漠-草原生态过渡带是监测全球气候变化和人类活动对生物影响的重要区域之一。本文以Sevilleta荒漠．草原生态过渡带为例,运用灰色系统分析方法．研究在当前全球气候变暖的条件下,1989～1998年10年间该生态过渡带的两种共存的植物优势种(Bouteloua eripoda)和(Bouteloua grncilis)历年的种群密度、组合形式和变化趋势,分析引起这些变化的主要气候因子。结果表明,Sevilleta生态过渡带中来自荒漠草原的优势物种Bouteloua eripoda的密度有上升的趋势,来自大草原的优势物种Bouteloua gracilis的密度有下降的趋势,它们的密度比大于1且有上升的趋势;在影响种群密度变化的降水、最高温度、最低温度、湿度的4个气候因子中,温度因子起着重要的作用。由此可以认为,随着气候的变暖,本区来自荒漠地区的优势植物种在荒漠-草原群落过渡带中将逐渐处于强优势地位,生态过渡带将有可能被荒漠草原所替代。     

Space,Time, Rhetoric and Agricultural Change in the Transition Zone of Ghana

This paper examines change within farming systems in the Brong Ahafo Region in Ghana, and the impact of agricultural modernization and mechanization on the regional economy and local farming systems. It combines anthropological, historical, and remote sensing techniques to document changes in farming practice and land use and land cover. It argues that change is not the product of simple evolutionary sequences of responses to population pressures or adoption of modern technologies, but arises out of a complex set of factors interacting within wider regional economies, which are increasingly commodified and commercialized and subject to global market pressures. These include technical, institutional, market, movements of labor, and transport infrastructure development dimensions, which often create new opportunities for local farmers other than those envisaged in agricultural development policies. Tracing the opening up of the transition zone over the last 40–50 years through the development of state farms and mechanized synthetic agriculture, the paper examines the changing fortunes of farming systems within a radius of 30–40 km from agricultural technology hubs and the implications for models of agricultural development.

Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens

Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo.     

Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance

Cilia are thought to harbour a membrane diffusion barrier within their transition zone (TZ) that compartmentalises signalling proteins. How this “ciliary gate” assembles and functions remains largely unknown. Contrary to current models, we present evidence that Caenorhabditis elegans MKS‐5 (orthologue of mammalian Mks5/Rpgrip1L/Nphp8 and Rpgrip1) may not be a simple structural scaffold for anchoring > 10 different proteins at the TZ, but instead, functions as an assembly factor. This activity is needed to form TZ ultrastructure, which comprises Y‐shaped axoneme‐to‐membrane connectors. Coiled‐coil and C2 domains within MKS‐5 enable TZ localisation and functional interactions with two TZ modules, consisting of Meckel syndrome (MKS) and nephronophthisis (NPHP) proteins. Discrete roles for these modules at basal body‐associated transition fibres and TZ explain their redundant functions in making essential membrane connections and thus sealing the ciliary compartment. Furthermore, MKS‐5 establishes a ciliary zone of exclusion (CIZE) at the TZ that confines signalling proteins, including GPCRs and NPHP‐2/inversin, to distal ciliary subdomains. The TZ/CIZE, potentially acting as a lipid gate, limits the abundance of the phosphoinositide PIP₂ within cilia and is required for cell signalling. Together, our findings suggest a new model for Mks5/Rpgrip1L in TZ assembly and function that is essential for establishing the ciliary signalling compartment.     

The perineurium of the adult housefly: Ultrastructure and permeability to lanthanum

Summary The ultrastructure of the perineurial cells of Musca overlying the first optic neuropile was examined by transmission electron microscopy. These cells are somewhat similar to those of other insects but cytoplasmic flanges seem to be absent, and mitochondria are relatively large and sinuous. The intercellular channel system on the lateral border of the cells is relatively spacious and highly meandering. Perineurial cells are joined by septate, gap, and tight junctions, hemidesmosomes, and desmosomes. Tight and septate junctions bond perineurial cells and glial cells. These data are evaluated on the basis of tracer studies with lanthanum. This material penetrates the extracellular space between perineurium and underlying glial and nerve cells, between epithelial glial cells and retinular axon terminals (capitate projections), and between the - fiber pair in the optic cartridge (gnarls). If no damage occurs to the perineurial cells during tissue preparation, this passage of lanthanum to neuronal surfaces indicates that the blood brain barrier is incomplete in this restricted area. Supportive evidence for such permeance is based on electrophysiological data, considerations of membrane specializations in the optic neuropile, and Na⁺/K⁺ ratios of dipteran hemolymph.We gratefully acknowledge support from the N.I.H., National Eye Institute, EYO 1686 and from the College of Agricultural and Life Sciences, Hatch Project 2100. Richard L. St. Marie and Professor Stanley D. Beck, Department of Entomology, UW, Madison read early drafts of this paper and provided constructive comments