Filamentous bodies in human glomerulonephritis

Summary Filamentous bodies have been identified in the glomerular cells of human kidney biopsies. These structures have a close morphological resemblance to ciliary rootlets, although the cells of the glomerular tuft only occasionally bear cilia. Their significance could be, as for cilia, of a cellular disdifferentiation of a pathological cellular proliferation.     

Mechanisms of ciliogenesis suppression in dividing cells

The primary cilium is a non-motile and microtubule-enriched protrusion ensheathed by plasma membrane. Primary cilia function as mechano/chemosensors and signaling hubs and their disorders predispose to a wide spectrum of human diseases. Most types of cells assemble their primary cilia in response to cellular quiescence, whereas they start to retract the primary cilia upon cell-cycle reentry. The retardation of ciliary resorption process has been shown to delay cell-cycle progression to the S or M phase after cell-cycle reentry. Apart from this conventional concept of ciliary disassembly linked to cell-cycle reentry, recent studies have led to a novel concept, suggesting that cells can suppress primary cilia assembly during cell proliferation. Accumulating evidence has also demonstrated the importance of Aurora-A (a protein originally identified as one of mitotic kinases) not only in ciliary resorption after cell-cycle reentry but also in the suppression of ciliogenesis in proliferating cells, whereas Aurora-A activators are clearly distinct in both phenomena. Here, we summarize the current knowledge of how cycling cells suppress ciliogenesis and compare it with mechanisms underlying ciliary resorption after cell-cycle reentry. We also discuss a reciprocal relationship between primary cilia and cell proliferation.     

Primary cilia proteins: ciliary and extraciliary sites and functions

Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these “ciliary” proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes’ influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of “cilia” proteins as a means to demonstrate the potential non-ciliary roles for these proteins.     

Mechanisms regulating cilia growth and cilia function in endothelial cells

The primary cilium is an important sensory organelle present in most mammalian cells. Our current studies aim at examining intracellular molecules that regulate cilia length and/or cilia function in vitro and ex vivo. For the first time, we show that intracellular cAMP and cAMP-dependent protein kinase (PKA) regulate both cilia length and function in vascular endothelial cells. Although calcium-dependent protein kinase modulates cilia length, it does not play a significant role in cilia function. Cilia length regulation also involves mitogen-activated protein kinase (MAPK), protein phosphatase-1 (PP-1), and cofilin. Furthermore, cofilin regulates cilia length through actin rearrangement. Overall, our study suggests that the molecular interactions between cilia function and length can be independent of one another. Although PKA regulates both cilia length and function, changes in cilia length by MAPK, PP-1, or cofilin do not have a direct correlation to changes in cilia function. We propose that cilia length and function are regulated by distinct, yet complex intertwined signaling pathways.     

Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet-Biedl syndrome proteins

Primary cilia are nearly ubiquitous cellular appendages that provide important sensory and signaling functions. Ciliary dysfunction underlies numerous human diseases, collectively termed ciliopathies. Primary cilia have distinct functions on different cell types and these functions are defined by the signaling proteins that localize to the ciliary membrane. Neurons throughout the mammalian brain possess primary cilia upon which certain G protein-coupled receptors localize. Yet, the precise signaling proteins present on the vast majority of neuronal cilia are unknown. Here, we report that dopamine receptor 1 (D1) localizes to cilia on mouse central neurons, thereby implicating neuronal cilia in dopamine signaling. Interestingly, ciliary localization of D1 is dynamic, and the receptor rapidly translocates to and from cilia in response to environmental cues. Notably, the translocation of D1 from cilia requires proteins mutated in the ciliopathy Bardet-Biedl syndrome (BBS), and we find that one of the BBS proteins, Bbs5, specifically interacts with D1.     

Neuronal ciliary signaling in homeostasis and disease

Primary cilia are a class of cilia that are typically solitary, immotile appendages present on nearly every mammalian cell type. Primary cilia are believed to perform specialized sensory and signaling functions that are important for normal development and cellular homeostasis. Indeed, primary cilia dysfunction is now linked to numerous human diseases and genetic disorders. Collectively, primary cilia disorders are termed as ciliopathies and present with a wide range of clinical features, including cystic kidney disease, retinal degeneration, obesity, polydactyly, anosmia, intellectual disability, and brain malformations. Although significant progress has been made in elucidating the functions of primary cilia on some cell types, the precise functions of most primary cilia remain unknown. This is particularly true for primary cilia on neurons throughout the mammalian brain. This review will introduce primary cilia and ciliary signaling pathways with a focus on neuronal cilia and their putative functions and roles in human diseases.     

Cell cycle progression by the repression of primary cilia formation in proliferating cells

In most cell types, primary cilia protrude from the cell surface and act as major hubs for cell signaling, cell differentiation, and cell polarity. With the exception of some cells ciliated during cell proliferation, most cells begin to disassemble their primary cilia at cell cycle re-entry. Although the role of primary cilia disassembly on cell cycle progression is still under debate, recent data have emerged to support the idea that primary cilia exert influence on cell cycle progression. In this review, we emphasize a non-mitotic role of Aurora-A not only in the ciliary resorption at cell cycle re-entry but also in continuous suppression of cilia regeneration during cell proliferation. We also summarize recent new findings indicating that forced induction/suppression of primary cilia can affect cell cycle progression, in particular the transition from G0/G1 to S phase. In addition, we speculate how (de)ciliation affects cell cycle progression.     

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.     

Routes and machinery of primary cilium biogenesis

Primary cilia are solitary, microtubule-based protrusions of the cell surface that play fundamental roles as photosensors, mechanosensors and biochemical sensors. Primary cilia dysfunction results in a long list of developmental and degenerative disorders that combine to give rise to a large spectrum of human diseases affecting almost any major body organ. Depending on the cell type, primary ciliogenesis is initiated intracellularly, as in fibroblasts, or at the cell surface, as in renal polarized epithelial cells. In this review, we have focused on the routes of primary ciliogenesis placing particular emphasis on the recently described pathway in renal polarized epithelial cells by which the midbody remnant resulting from a previous cell division event enables the centrosome for initiation of primary cilium assembly. The protein machinery implicated in primary cilium formation in epithelial cells, including the machinery best known for its involvement in establishing cell polarity and polarized membrane trafficking, is also discussed.     

Cilia in stellate neurons of the rat cerebellum

Summary Cilia in stellate neurons of the normal rat cerebellum are described. 8 cilia were observed in a total of 60 cells studied. An 8+1 pattern was found throughout their length. Furthermore, no arms, spokes or other accessory structures necessary for ciliary motion were seen. These findings make it possible to suggest that these cilia are probably without function and are related to the epithelial origin of these cells.This work was granted by INIC: Centro de Morfologia Experimental (MbP₁).Acknowledgments. The authors thank M.A. Rodrigues, M. M. Pacheco, M. C.Pinho and L. B. Nunes for technical assistance.     

The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis

Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They have been implicated, together with their interacting minus-end directed motor protein KIFC5A, in the regulation of centriole duplication in mammalian cells. Here we show that Nubp1 and Nubp2 are integral components of centrioles throughout the cell cycle, recruited independently of KIFC5A. We further demonstrate their localization at the basal body of the primary cilium in quiescent vertebrate cells or invertebrate sensory cilia, as well as in the motile cilia of mouse cells and in the flagella of Chlamydomonas. RNAi-mediated silencing of nubp-1 in C. elegans causes the formation of morphologically aberrant and additional cilia in sensory neurons. Correspondingly, downregulation of Nubp1 or Nubp2 in mouse quiescent NIH 3T3 cells markedly increases the number of ciliated cells, while knockdown of KIFC5A dramatically reduces ciliogenesis. Simultaneous double silencing of Nubp1 + KIFC5A restores the percentage of ciliated cells to control levels. We document the normal ciliary recruitment, during these silencing regimes, of basal body proteins critical for ciliogenesis, namely CP110, CEP290, cenexin, Chibby, AurA, Rab8, and BBS7. Interestingly, we uncover novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which we find enriched at the basal body and recruited independently of the Nubps or KIFC5A. Our combined results for Nubp1, Nubp2, and KIFC5A and their striking effects on cilium formation suggest a central regulatory role for these proteins, likely involving CCT/TRiC chaperone activity, in ciliogenesis.     

The primary cilium as a dual sensor of mechanochemical signals in chondrocytes

The primary cilium is an immotile, solitary, and microtubule-based structure that projects from cell surfaces into the extracellular environment. The primary cilium functions as a dual sensor, as mechanosensors and chemosensors. The primary cilia coordinate several essential cell signaling pathways that are mainly involved in cell division and differentiation. A primary cilium malfunction can result in several human diseases. Mechanical loading is sense by mechanosensitive cells in nearly all tissues and organs. With this sensation, the mechanical signal is further transduced into biochemical signals involving pathways such as Akt, PKA, FAK, ERK, and MAPK. In this review, we focus on the fundamental functional and structural features of primary cilia in chondrocytes and chondrogenic cells.     

Surface morphology of the subfornical organ: effects of low and high sodium chloride diet

Ependymal cells found in the subfornical organ of the rat were counted. Cells covered by small microvilli, small protrusions and smooth cells were frequently found. Also present were cells with long or short cilia, cels with large protrusions and supraependymal cells. High and low sodium diets reduced the number of cells with large protrusions. Microvilli-covered cells increased after a low sodium diet.     

Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 ^?/? cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.     

Ultrastructural investigation of fetal rat brain hemisphere tissue in nonadherent stationary organ culture

Fetal rat brain fragments grown in nonadherent stationary organ culture for 50 days have been investigated ultrastructurally. Synaptogenesis and myelin formation occurred at the same time as the corresponding time-dependent events in the developing brain in vivo. Intermediate junctions were observed between cellular processes lining a central cavity in the fragments and later associated with astrocytes at the surface. Gap junctions and tight junctions were also present. In some fragments cilia were observed in the central cavity. Subependymal basement membrane labyrinths were observed in all fragments after 10 days in culture. The ultrastructural characteristics and the tissue-like structure in general were preserved for at least 50 days in this tissue culture system. The brain fragments may therefore be a valuable supplement to existing culture methods for nervous tissue.     

Über die Bildung rudimentärer Zilien bei Melanozyten

Summary The centriols of the melanocytes and malignant melanoma cells appear as diplosome and show the well-known substructure. The formation of rudimentary cilia in single benign human pigmented tumor cells was observed. These findings are discussed in relation to the origin of the melanocytes.     

Fibrin membrane endowed with biological function. III. Fixing living cells in fibrin gel without impairing their functions

Summary Chlorella cells, sea urchin eggs and Paramecium were embedded in fibrin gel which was formed by fibrinogen-fibrin conversion in the presence of thrombin. The embedded Chlorella cells retain the ability of photosynthesis by illumination. The embedded sea urchin eggs develop to normal blastulae and gastrulae. Samples of Paramecium survive for more than several h beating their cilia. It is suggested that this technique of fixing living cells is useful for handling free cells as a mass like a tissue, and for holding free cells in micrurgical experiments.     

Ultrastructural investigation of fetal rat brain hemisphere tissue in nonadherent stationary organ culture

Summary Fetal rat brain fragments grown in nonadherent stationary organ culture for 50 days have been investigated ultrastructurally. Synaptogenesis and myelin formation occurred at the same time as the corresponding time-dependent events in the developing brain in vivo. Intermediate junctions were observed between cellular processes lining a central cavity in the fragments and later associated with astrocytes at the surface. Gap junctions and tight junctions were also present. In some fragments cilia were observed in the central cavity. Subependymal basement membrane labyrinths were observed in all fragments after 10 days in culture. The ultrastructural characteristics and the tissue-like structure in general were preserved for at least 50 days in this tissue culture system. The brain fragments may therefore be a valuable supplement to existing culture methods for nervous tissue.Research fellow from the Norwegian Research Council for Sciences and Humanities. This work was supported by the Norwegian Cancer Society.     

Ciliated fibroblasts and smooth muscle cells in the rat uterus

Summary Ciliation in endometrial fibroblasts and myometrial muscle cells of the rat was examined by transmission electron microscopy. Quantification of the number of ciliated cells during the estrus cycle did not show any firm relationship between cilation and ovarian hormonal activity. In the case of most cilia, there is a spatial relationship between their basal centrioles and the Golgi complex, so that a Golgi-cilium complex is created. A possible role of ciliation in uterine fibroblasts and smooth muscle cells is discussed.