Paddle cilia (discocilia) in chemosensitive structures of the gastropod mollusk Pleurobranchaea californica

Summary Scanning electron microscopy of various regions of the body of the marine gastropod Pleurobranchaea californica (McFarland) has revealed a characteristic cell type that bears cilia with dilated discoid-shaped tips. The tips of the cilia consist of an expansion of the ciliary membrane around a looped distal extension of the axoneme. These kinocilia have been observed in numerous other marine invertebrates and are generally referred to as paddle cilia (Tamarin et al. 1974) or discocilia (Heimler 1978). Although many functions have been proposed for paddle cilia, little empirical evidence supports any of the proposals. In Pleurobranchaea we have found that the distribution of this ciliated cell type corresponds exactly to areas of the body known from behavioral studies (Lee et al. 1974; Davis and Matera 1981) to mediate chemoreception. Transmission electron microscopy of the epithelium lining the rhinophores and tentacles of Pleurobranchaea revealed details of the ultrastructure of these ciliated cells and showed that they are primary receptors. These ciliated receptors lie in a yellow-brown pseudostratified columnar epithelium that superficially resembles the olfactory mucosa of vertebrates.     

Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

Primary cilia disappear in rat podocytes during glomerular development

Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88?±?5%, n?=?3), capillary loop (95?±?4%, n?=? 4), and maturing glomerulus (76?±?13%, n?=?5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0?±?0%, n?=?11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes.     

Scanning electron microscopy of olfactory rosette in sea trout

Summary Scanning electron microscopy has been employed to study the central axis and laminae of the olfactory rosette in adult sea trout (Salmo trutta trutta L.) caught in the River Umeälven when they were homing from sea.—Both flat sides of the primary laminae are secondarily folded all over their surface. In one organ there are about 200 secondary laminae usually arranged in longitudinal, parallel ridges crossing the surface of the primary laminae. Initially they are covered with sensory epithelium, but as the folds grow they become covered with an increasing area of indifferent ciliar epithelium with bushes of cilia separated by microvilli cells and goblet cells. Parts of the central axis and primary laminae have a nonciliar indifferent epithelium. The sensory epithelium has irregularly arranged cilia. Like those of the indifferent epithelium they have uniform thickness and granulated surface. The function of laminae, secretion and cilia is discussed.The author wish to acknowledge the technical facilities and assistance in the use of the scanning electron microscope to Jeolco Stockholm office. This research was supported by grants 2389-10, 2389-11 and 2389-13 from the Swedish Natural Science Research Council.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

Ultrastructure of epidermal eyespots of Microstomum lineare (Turbellaria,macrostomida)

Summary The eyespots of Microstomum lineare were studied by electron microscopy, light microscopy, and fluorescence microscopy. Each eyespot consists of two ciliary photoreceptor cells shielded by pigment cells and additional sensory cells. The photoreceptor cells are characterized by a distal intracellular cavity lined with 50–100 interwoven cilia. The other sensory cells are of two ultrastructurally different types, one with long cilia predominating and the other with balloonlike cilia. The pigment cells, which envelop processes of the sensory cells, contain pigment vacuoles varying in size and content and give a bright red fluorescence by the Falck-Hillarp method. The eyespots are suggested to perform a dual function as photoreceptors and chemoreceptors. The evolutionary significance of ciliary photoreceptors in Turbellaria is discussed.     

Ciliated Epidermal Cells in Non-teleost Actinopterygian Fish

Scanning electron microscopy reveals the presence of ciliated epidermal cells that form halos around canal pores and pit lines of the mechanoreceptive lateral line system in two actinopterygian fish, Polypterus and Acipenser. Transmission electron microscopy shows that the cilia exhibit a typical 9 + 2 microtubule configuration and are not directionally polarized. The function, developmental origin and systematic significance of ciliated epidermal cells in actinopterygian fish are considered.     

The Leber Congenital Amaurosis Protein AIPL1 and EB Proteins Co-Localize at the Photoreceptor Cilium

Purpose

The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).

Methods

The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.

Results

Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.

Conclusions

AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.     

Ultrastructure of the epidermis of adult and embryonic Paravortex species (Turbellaria,Eulecithophora)

The epidermis and associated structures of adult and embryonic Paravortex cardii and Paravortex karlingi, internal parasites of Cerastoderma edule, have been examined using scanning and transmission electron microscopy. The cellular epidermis of adult Paravortex bears cilia and microvilli which differ in number and distribution between P. karlingi and P. cardii. Cellular organelles include mitochondria, lipid bodies, Golgi bodies, and ultrarhabdites. Epidermal nuclei are located in the proximal portion of the cells. The development of the tegument of embryo Paravortex has been described and a possible origin for the embryo capsule is suggested. These findings are discussed in relation to the phylogenetic status of the Turbellaria in relation to other Platyhelminthes and in the functional adaptation of the epidermis for a parasitic mode of life.Abbreviations bb- basal bodies - bl- basal lamella - c- cilia - cp- capsule - dc- dark cells - e- embryos - ep- epidermis - g- Golgi bodies - int- interdigitation (of cells) - l- lipid - lf- lamellar fold - mc- migrating cell - mf- membranous folds - mt- mitochondria - mv- microvilli - n- nucleus - nb- neoblasts - p- projections of epidermis - par- parenchyma of mother - pr- primary rootlet - rc- rhabditogen cells - sr- secondary rootlet - ur- ultrarhabdites - vt- vitelline material     

Embryonic expression of pericentrin suggests universal roles in ciliogenesis

Pericentrin (Pcnt) is a giant coiled-coil protein known to mediate microtubule organization. It has been recently reported that mitosis-specific centrosomal anchoring of γ tubulin complexes by Pcnt acts to control mitotic spindle organization, though little is known about the in vivo expression of Pcnt. In this study, we investigated Pcnt expression in mouse embryos. In situ hybridization analysis revealed preferential expression of Pcnt in quiescent G₀ phase cells throughout the embryo with an unexpectedly low expression level in proliferating cells, suggesting that Pcnt might not play an important role in mitotic proliferation. Immunofluorescence analysis confirmed preferential expression of the Pcnt protein in G₀ phase cells. Moreover, Pcnt was shown to be localized to the base of primary cilia in multiple embryonic tissues, in agreement with a recent study demonstrating the involvement of Pcnt in primary cilia formation using cultured mammalian cells.     

Endothelial primary cilia inhibit atherosclerosis

Primary cilia are microtubule‐based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe^?/? mice fed a high‐fat, high‐cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro‐atherosclerotic signaling in the aorta.     

Secondary Messengers in the Locomotor-Sensory System of Early Multicellulars. Inositol-Containing Compartments in Receptor Cells of Ctenophores and Coelenterates according to the Data of Cytochemical Investigation

The electron microscopy cytochemical data have been obtained on compartmentalization of a secondary messenger, inositol (1,4,5)-triphosphate (InsP₃), in receptor cells of different modalities in representatives of early multicellular animals, ctenophores Bero cucumis and coelenterates, scyphomedusas Cyanea arctica. In the gravitation mechanoreceptor cells of the ctenophores and medusas, the inositol-containing compartments were revealed predominantly in cilia and stereocilia. The presence of uniformly distributed discrete granules of the precipitate of the enzymatic cytochemical reaction product in the light-sensitive lamellar structures of photoreceptor cells of ctenophores is discussed in the connection with the possible existence of peculiar rhodopsin–inositol complexes participating in photoreceptor processes. The peculiarities of compartmentalization of the precipitate in photo- and gravitation mechanoreceptor cells allow considering InsP₃ as one of the components that that are able to contribute to development of mechanisms of modality in the ciliary receptor cells of ancient multicellular animals.     

赤霉素对紫斑牡丹种子下胚轴萌发及其种皮和胚乳结构的影响

以紫斑牡丹种子为试验材料,考察不同浓度赤霉素(GA_3)处理下种子生根情况,同时对其种皮纹饰进行了扫描电镜(SEM)观察,并对胚乳结构进行了荧光倒置显微镜、透射电镜(TEM)观察,以揭示GA_3对紫斑牡丹种子下胚轴解除休眠过程中种子种皮、胚乳结构变化和生根的影响。结果表明:(1)GA_3处理能够促进紫斑牡丹种子生根,以300 mg/L GA_3处理的种子生根效果最好,与对照相比可提前14.66 d生根,生根率可达62.33%。(2)扫描电镜观察发现,紫斑牡丹种皮表面含有蜡质,种子萌发进程中蜡质明显不断增加,种皮表面网眼逐渐消失,种皮皱缩明显。(3)光学显微镜观察发现,GA_3处理的紫斑牡丹种子胚乳细胞中含有较多淀粉体和蛋白体,并随着种子萌发进程逐渐积累,可为种子萌发提供足够的养分。(4)透射电镜观察发现,GA_3处理的紫斑牡丹种子胚乳细胞内大分子营养物质不断降解,线粒体、内质网、高尔基体等细胞器相继出现,种子下胚轴休眠解除,细胞壁较薄后期消融,利于营养物质传送。研究认为,适宜浓度GA_3处理可有效加快紫斑牡丹种皮结构的改变和胚乳细胞内物质代谢进程,促进种子萌发,且300 mg/L GA_3处理紫斑牡丹种子24 h可以获得最好的生根效果。     

A re-description of the ciliate genus and type species, Balantidium entozoon

Members of the ciliate genus Balantidium possess a specialized “Villeneuve-Brachon's” field of somatic cilia to the right of the vestibule, or in a dextroral location. Specimens of the type species were collected in Italy and fixed for study by light microscopy, and scanning and transmission electron microscopy. The presence of the field in the type species and several other species of the genus indicates a need to re-describe the genus by including details of the ultrastructure of that field. Scanning electron microscopy shows that the field consists of one row of relatively short cilia of uniform length flanked on each side by 2–3 rows, or more, of very short cilia. Their kinetids have typical litostome structure in transmission electron micrographs. We speculate on a possible function for the Villeneuve-Brachon's field and also present morphometric data on the type species. The base sequence of the small subunit ribosomal RNA gene of Balantidium entozoon has been determined and found to differ by 5% from that of B. coli. Based on the location and ultrastructure, organelles found around the somatic kinetosomes and within inter-kinetal ridges of B. entozoon were identified as hydrogenosomes.     

Evolutionary implications of localization of the signaling scaffold protein Parafusin to both cilia and the nucleus

Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) – as we proved by comparison of their 88 sequences – but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS‐specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT‐2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.     

Spinous extensions on ciliary necklaces in ependymal cells

Summary In ependymal cells of the mouse the neck region of all cilia examined by means of transmission electron microscopy exhibited rows of electron-dense spines. These structures correspond to the ciliary necklace reported from freeze-etch studies, a structure presumed to serve as an energy-regulating system in motile cilia. Send offprint requests to: Institute of Neuropathology, Free University Berlin, Hindenburgdamm 30, 1 Berlin 45     

The Olfactory System in the Hagfish Myxine glutinosa

The apical part of the olfactory epithelium in Myxine glutinosa was investigated by optical and electron microscopy. This part of the epithelium consists of supporting cells and two types of olfactory receptor cells, i.e., ciliated receptor cells and microvillous receptor cells. The olfactory cilia have a 9 + 0 pattern of the microtubules, occasionally with one pair of the doublets dislocated towards the center of the cilium. Giant cilia were observed. The supporting cells bear microvilli and are rich in tonofilaments. The supporting cells also have a secretory function, their secretion consisting mainly of acid mucopolysaccharides. An asymmetrical type of desmosome was found between the olfactory receptor cells and the supporting cells.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.