The active and passive ciliary motion in the embryo node: A computational fluid dynamics model

The breaking of left–right symmetry in the mammalian embryo is believed to occur in a transient embryonic structure, the node, when cilia create a leftward flow of liquid. The two-cilia hypothesis proposes that the node contains two kinds of primary cilia: motile cilia that rotate autonomously to generate the leftward fluid flow and passive cilia that act as mechano-sensors, responding to flow. While studies support this hypothesis, the mechanism by which the sensory cilia respond to the fluid flow is still unclear. In this paper, we present a computational model of two cilia, one active and one passive. By employing computational fluid dynamics, deformable mesh computational techniques and fluid–structure interaction analysis, and solving the three-dimensional unsteady transport equations, we study the flow pattern produced by the movement of the active cilium and the response of the passive cilium to this flow. Our results reveal that clockwise rotation of the active cilium can generate a counter-clockwise elliptical rotation and overall lateral displacement for its neighboring passive one, of measurable magnitude and consistent pattern. This supports the plausibility of the two-cilia hypothesis and helps quantify the motion pattern for the passive cilium induced by this regional flow.     

Cilium: Origin and 9-fold symmetry

How the cilium appeared is still such a poorly defined question that current hypotheses range from a symbiotic spirochaete to a cellular eye. In this paper, the whole question is subdivided into a list of problems which are morphological, physiological and temporal. These problems are examined one by one, in order to analyse the most popular exogenous hypothesis of Margulis as well as other recent exogenous and endogenous hypotheses. To overcome fundamental topological and temporal difficulties, a new endogenous hypothesis is expounded, according to which the cilium derives from a cellular peduncle reinforced with microtubules. This hypothesis implies a geometrical rationale for the ninefold symmetry. In the last paragraph the consequences of the various hypotheses are compared.     

Ultrastructural basis of the coordination of ciliary movement. Contractile function of the interconnecting cross-striated roots in ependymal epithelium

A hypothesis for contraction was suggested (molecular basis of the transmission of ciliary coordination) by means of the slipping and interdigitation of the microfilaments of one period with those of the adjacent periods, occurring at the level of the periodic band. The amplitude of the period is greater in the relaxed than in the contracted state, while the reverse is true with the periodic band amplitude. The microfilament length is constant, probably varying with the species. This hypothesis of the slipping and interdigitation of the microfilaments would be the basis of the ciliary coordination from cilium to cilium and from cell to cell, because of the anchoring of the bunches in the peribasal complexes and in the adherent connection complexes, respectively.     

Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation

Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10⁻¹² N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10⁻¹⁰ N/rad²; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.     

Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope.

The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.     

THE FINE STRUCTURE OF COCKROACH CAMPANIFORM SENSILLA

Campaniform sensilla on cockroach legs provide a good model system for the study of mechanoreceptive sensory transduction. This paper describes the structure of campaniform sensilla on the cockroach tibia as revealed by light- and electron-microscopy. Campaniform sensilla are proprioceptive mechanoreceptors associated with the exoskeleton. The function of each sensillum centers around a single primary sense cell, a large bipolar neuron whose 40 µ-wide cell body is available for electrophysiological investigation with intracellular microelectrodes. Its axon travels to the central nervous system; its dendrite gives rise to a modified cilium which is associated with the cuticle. The tip of the 20 µ-long dendrite contains a basal body, from which arises a 9 + 0 connecting cilium. This cilium passes through a canal in the cuticle, and expands in diameter to become the sensory process, a membrane-limited bundle of 350–1000 parallel microtubules. The tip of the sensory process is firmly attached to a thin cap of exocuticle; mechanical depression of this cap, which probably occurs during walking movements, effectively stimulates the sensillum. The hypothesis is presented that the microtubules of the sensory process play an important role in mechanoelectric transduction in cockroach campaniform sensilla.     

The mechanics of the primary cilium: an intricate structure with complex function

The primary cilium is a non-motile singular cellular structure that extends from the surface of nearly every cell in the body. The cilium has been shown to play numerous roles in maintaining tissue homeostasis, through regulating signaling pathways and sensing both biophysical and biochemical changes in the extracellular environment. The structural performance of the cilium is paramount to its function as defective cilia have been linked to numerous pathologies. In particular, the cilium has demonstrated a mechanosensory role in tissues such as the kidney, liver, endothelium and bone, where cilium deflection under mechanical loading triggers a cellular response. Understanding of how cilium structure and subsequent mechanical behavior contributes to the roles that cilium plays in regulating cellular behavior is a compelling question, yet is a relatively untouched research area. Recent advances in biophysical measurements have demonstrated the cilium to be a structurally intricate organelle containing an array of load bearing proteins. Furthermore advances in modeling of this organelle have revealed the importance of these proteins at regulating the cilium's mechanosensitivity. Remarkably, the cilium is capable of adapting its mechanical state, altering its length and possibly it's bending resistance, to regulate its mechanosensitivity demonstrating the importance of cilium mechanics in cellular responses. In this review, we introduce the cilium as a mechanosensor; discuss the advances in the mechanical modeling of cilia; explore the structural features of the cilium, which contribute to its mechanics and finish with possible mechanisms in which alteration in structure may affect ciliary mechanics, consequently affecting ciliary based mechanosensing.     

Morphological Aspects of Ciliary Motility

In Elliptio complanatus lateral cilia, two distinct patterns of filament termination can be discerned. In one case, all nine filaments are present and all are single; in the second, at least one filament is missing but doublets are still present. These probably represent different configurations within one cilium in different stroke positions; to get from one to the other, some peripheral filaments must move with respect to others. The data are consistent with the hypothesis that the filaments themselves do not change length, but rather slide past one another to accommodate increasing curvature. The bent regions of the cilium are in the form of circular arcs. In a few cases, apparent displacement of filaments at the tip (Δl) can be shown to be accounted for if we assume that all differences are generated within these arcs. The displacement per degree of bend is 35 A. Regions of bent arc are initially confined to the base of the cilium but move up the shaft as straight regions appear below them. From the relationship between arc length and radius of curvature, a shaft length that is the unit that initially bends and slides may be defined. Quantal displacements of the length of one 14S dynein may perhaps occur at sites between filaments at opposite sides of such a unit as sliding occurs.     

Removal of the MDCK Cell Primary Cilium Abolishes Flow Sensing

The hypothesis that cell primary cilium is solely responsible for the flow-induced Ca2+ response in MDCK cells was tested by removal of the cilia from mature, responsive cells. Incubation of the cells with 4 mM chloral hydrate for 68 hours resulted in the complete loss of the primary cilia and in disorganization of microtubules, as visualized by immunofluorescence. When intracellular Ca2+ concentration was measured with Fluo-4, the elevation that normally accompanies an increase in fluid flow was abolished after 20 hours exposure to chloral hydrate. At this time, the primary cilia still remained attached to the cells but had become twisted and flexible. Twenty-four hours after return of the deciliated cells to normal medium, intracellular microtubule organization appeared normal, but primary cilia had not yet been expressed. The cells failed to increase intracellular Ca2+ in response to fluid flow until after they had been in normal medium for 120 hours, at which time the primary cilia were 3-4 microm long. Chloral hydrate did not impair the Ca2+ mobilization machinery, as the Ca2+ response to mechanical contact and the spread to neighboring cells was unaffected by the drug. We conclude that the primary cilium is the only sensor for the flow-induced Ca2+ response in MDCK cells and estimate that a single mechanically sensitive channel in the cilium could provide the requisite Ca2+ influx.     

Dynamics of the primary cilium in shear flow

In this work, the equilibrium shape and dynamics of a primary cilium under flow are investigated by using both theoretical modeling and experiment. The cilium is modeled as an elastic beam that may undergo large deflection due to the hydrodynamic load. Equilibrium results show that the anchoring effects of the basal body on the cilium axoneme behave as a nonlinear rotational spring. Details of the rotational spring are elucidated by coupling the elastic beam with an elastic shell. We further study the dynamics of cilium under shear flow with the cilium base angle determined from the nonlinear rotational spring, and obtain good agreement in cilium bending and relaxing dynamics when comparing between modeling and experimental results. These results potentially shed light on the physics underlying the mechanosensitive ion channel transport through the ciliary membrane.     

The PCM–basal body/primary cilium coalition

The centrosome is an organelle that acts as a microtubule-organizing center (MTOC) throughout the cell cycle. Within the centrosome are often two components that each have an ability to organize microtubule structures: the centriole that has the potential to function as a basal body and nucleate a cilium or a flagellum and a mass of protein material that in the presence of a centriole is commonly referred to as the pericentriolar material (PCM) that organizes cytoplasmic and spindle microtubule arrays. One characteristic of a large variety of cells is the ability to express a non-motile primary cilium. It is now appreciated that the function of the primary cilium is integral to a variety of essential cell functions and defects affecting this structure underlie a variety of human disease. While the function of the primary cilium and manner in which a basal body organizes a primary cilium has received extensive attention there is now a need to explore the inter-relationship between the PCM and the basal body/primary cilium. It is this latter topic that is the focus of this review where we show that the PCM is integrated with the centriole to form a coalition that is essential for both the expression and function of the primary cilium as well as the organization and function of the cellular environment that surrounds it.     

Cell shape and contractility regulate ciliogenesis in cell cycle-arrested cells

In most lineages, cell cycle exit is correlated with the growth of a primary cilium. We analyzed cell cycle exit and ciliogenesis in human retinal cells and found that, contrary to the classical view, not all cells exiting the cell division cycle generate a primary cilium. Using adhesive micropatterns to control individual cell spreading, we demonstrate that cell spatial confinement is a major regulator of ciliogenesis. When spatially confined, cells assemble a contractile actin network along their ventral surface and a protrusive network along their dorsal surface. The nucleus-centrosome axis in confined cells is oriented toward the dorsal surface where the primary cilium is formed. In contrast, highly spread cells assemble mostly contractile actin bundles. The nucleus-centrosome axis of spread cells is oriented toward the ventral surface, where contractility prevented primary cilium growth. These results indicate that cell geometrical confinement affects cell polarity via the modulation of actin network architecture and thereby regulates basal body positioning and primary cilium growth.     

Electron microscope observations on the submicroscopic organization of the retinal rods

The submicroscopic organization of the retinal rods of the rabbit has been studied with high resolution electron microscopy in thin longitudinal and cross-sections. The outer rod segment consists of a stack of flattened sacs or cisternae each of them limited by a thin homogeneous membrane of about 30 A. The membrane of the rod sacs is attached to the surface membrane and is also in continuity with short tubular stalks of about 100 to 150 A which apparently end in relation with the connecting cilium. The bundle of filaments that constitute the connection between the outer and the inner segments is described under the name of connecting cilium. This fibrous component has a structure that is very similar to that of the cilium. It shows 9 pairs of peripheral filaments of about 160 A in diameter, a matrix material, and a surface membrane. Very infrequently two central single filaments are observed. The connecting cilium has a typical basal body in the inner segment; its distal end penetrates the outer segment, where it establishes some structural relation to the rod sacs. The relationships and submicroscopic organization of the connecting cilium were studied in longitudinal and in cross-sections passing at different levels of the rod segments. The inner rod segment shows two distinct regions: a distal and a proximal one. The distal region, corresponding to the ellipsoid of classical histology is mainly composed of longitudinally packed mitochondria. It also contains the basal body of the cilium, vacuoles of the endoplasmic reticulum, dense particles, and intervening matrix with very fine filaments. In the proximal region of the inner segment the mitochondria are lacking and within the matrix it is possible to recognize elements of the Golgi complex, vacuoles of the endoplasmic reticulum, dense particles and numerous neuroprotofibrils of 160 to 200 A in diameter which collect and form a definite bundle at the exit of the rod fiber. The interpretation of the connecting fibers as a portion of a cilium and of the outer segment as a differentiation of the distal part of a primitive cilium are discussed. The importance of the continuity of the surface membranes of the outer segment, connecting cilium, and inner segment is emphasized and its possible physiological role is discussed.     

Wnt signalling escapes to cilia

The primary cilium is proposed to restrain the level of canonical Wnt signalling, but it was unknown how the cilium achieves this. β-catenin, a component of the canonical Wnt signalling pathway, is now shown to be sequestered to the cilium by the Wnt signalling modulator Jouberin (Jbn) to restrain Wnt responses.     

Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis

Serial ultrathin sections were used to study the formation of the primary cilium and the centriolar apparatus, basal body, and centriole in the neuroepithelial primordial cell of the embryonic nervous system in the mouse. At the end of mitosis, the centrioles seem to migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. One of these centrioles, the nearest to the ventricular surface, begins to mature to form a basal body, since its tip is capped by a vesicle probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growth by the centrifugal extension of the 9 sets of microtubule doublets. These 9 sets invade the thick base of the cilium which is initially capped by a ball-shaped tip with the appearance of a mushroom cilium. The secondary extension of 7, then 5, and finally 2 sets of microtubule doublets contribute to form the tip of the mature cilium, which is associated with a mature centriolar apparatus formed by a basal body and a centriole. Centriologenesis occurs before mitosis and is concomitant with the progressive resorption of the cilium. The daughter centriole, or procentriole, begins to take form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the microtubules of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the primordial cells of the embryonic nervous system, centriologenesis and ciliogenesis are 2 independent phenomena.     

In vitro investigation of renal epithelial injury suggests that primary cilium length is regulated by hypoxia-inducible mechanisms

Primary cilia are non-motile sensory organelles that project from cells in many tissues. The role of renal primary cilium-based signalling in regulating epithelial cell proliferation and differentiation is highlighted by studies showing that defects of the cilium lead to epithelial de-differentiation, over proliferation and polycystic kidney disease. Recent studies show that renal primary cilia may also play a role in controlling epithelial differentiation during renal repair. After injury, renal cilium length increases dramatically and then undergoes a normalization that coincides with structural and functional repair in both human patients and mouse models of renal injury. These changes in cilium length are likely to modulate cilium-based signalling, but the injury-related factors that influence renal primary cilium length have yet to be determined. Here, we investigated the effect of three factors commonly associated with renal injury on renal cilium length in an in vitro setting. MDCK (Madin Darby canine kidney) cell cultures bearing primary cilia were treated with BSA to simulate albuminuria, cobalt chloride to simulate hypoxia and the inflammation-related cytokine tumour necrosis factor α. Primary cilium length was only increased in cultures treated with cobalt chloride. Our results suggest a role for hypoxia and the induction of HIF-1α (hypoxia-inducible factor 1α) in increasing renal primary cilium length following renal injury.     

Enteric neurons show a primary cilium

The primary cilium is a non‐motile cilium whose structure is 9+0. It is involved in co‐ordinating cellular signal transduction pathways, developmental processes and tissue homeostasis. Defects in the structure or function of the primary cilium underlie numerous human diseases, collectively termed ciliopathies. The presence of single cilia in the central nervous system (CNS) is well documented, including some choroid plexus cells, neural stem cells, neurons and astrocytes, but the presence of primary cilia in differentiated neurons of the enteric nervous system (ENS) has not yet been described in mammals to the best of our knowledge. The enteric nervous system closely resembles the central nervous system. In fact, the ultrastructure of the ENS is more similar to the CNS ultrastructure than to the rest of the peripheral nervous system. This research work describes for the first time the ultrastructural characteristics of the single cilium in neurons of rat duodenum myenteric plexus, and reviews the cilium function in the CNS to propose the possible role of cilia in the ENS cells.     

Observations on the Caudal Cilium of the Tomite of Ichthyophthirius multifiliis Fouquet, 18761,2

Light microscopy of live or silver-impregnated specimens of the fish parasite Ichthyophthirius multifiliis show that the tomites are elongated and claviform with the anterior end broad. The cytostome, indicated by the presence of the organelle of Lieberkühn, is found in the lower part of the broadened anterior third of the tomite. The tapered posterior end bears a rigid, caudal cilium at its pole. Scanning electron microscopy reveals the caudal cilium and associated structures, including the depression from which the cilium protrudes, circumciliary ring, and raised struts on the ring. From these observations it is concluded that a previously reported “apical filament” found on the tomite is actually the posterior caudal cilium described by Canella & Rocchi-Canella in 1976.     

Vertebrate primary cilia: a sensory part of centrosomal complex in tissue cells,but a “sleeping beauty” in cultured cells?

Primary cilium development along with other components of the centrosome in mammalian cells was analysed ultrastructurally and by immunofluorescent staining with anti-acetylated tubulin antibodies. We categorized two types of primary cilia, nascent cilia that are about 1microm long located inside the cytoplasm, and true primary cilia that are several microm long and protrude from the plasma membrane. The primary cilium is invariably associated with the older centriole of each diplosome, having appendages at the distal end and pericentriolar satellites with cytoplasmic microtubules emanating from them. Only one cilium per cell is formed normally through G(0), S and G(2)phases. However, in some mouse embryo fibroblasts with two mature centrioles, bicilates were seen. Primary cilia were not observed in cultured cells where the mature centriole had no satellites and appendages (Chinese hamster kidney cells, line 237, some clones of l-fibroblasts). In contrast to primary cilia, striated rootlets were found around active and non-active centrioles with the same frequency. In proliferating cultured cells, a primary cilium can be formed several hours after mitosis, in fibroblasts 2-4 h after cell division and in PK cells only during the S-phase. In interphase cells, formation of the primary cilium can be stimulated by the action of metabolic inhibitors and by reversed depolymerization of cytoplasmic microtubules with cold or colcemid treatments. In mouse renal epithelial cells in situ, the centrosome was located near the cell surface and mature centrioles in 80% of the cells had primary cilium protruding into the duct lumen. After cells were explanted and subcultured, the centrosome comes closer to the nucleus and the primary cilium was depolymerized or reduced. Later primary cilia appeared in cells that form islets on the coverslip. However, the centrosome in cultured ciliated cells was always located near the cell nucleus and primary cilium never formed a characteristic distal bulb. A sequence of the developmental stages of the primary cilium is proposed and discussed. We also conclude that functioning primary cilium does not necessarily operate in culture cells, which might explain some of the contradictory data on cell ciliation in vitro reported in the literature.