Structure and function of mammalian cilia

In the past half century, beginning with electron microscopic studies of 9 + 2 motile and 9 + 0 primary cilia, novel insights have been obtained regarding the structure and function of mammalian cilia. All cilia can now be viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation. This view has had unanticipated consequences for our understanding of developmental processes and human disease

A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling

The serologically defined colon cancer antigen-3 (SDCCAG3) is involved in the regulation of ciliogenesis

A primary cilium is present on most eukaryotic cells and represents a specialized organelle dedicated to signal transduction and mechanosensing. Defects in cilia function are the cause for several human diseases called ciliopathies. The serologically defined colon cancer antigen-3 (SDCCAG3) is a recently described novel endosomal protein mainly localized at early and recycling endosomes and interacting with several components of membrane trafficking pathways. Here we describe localization of SDCCAG3 to the basal body of primary cilia. Furthermore, we demonstrate that decreased expression levels of SDCCAG3 correlate with decreased ciliary length and a reduced percentage of ciliated cells. We show that SDCCAG3 interacts with the intraflagellar transport protein 88 (IFT88), a crucial component of ciliogenesis and intraciliary transport. Mapping experiments revealed that the N-terminus of SDCCAG3 mediates this interaction by binding to a region within IFT88 comprising several tetratricopeptide (TRP) repeats. Finally, we demonstrate that SDCCAG3 is important for ciliary localization of the membrane protein Polycystin-2, a protein playing an important role in the formation of polycystic kidney disease, but not for Rab8 another ciliary protein. Together these data suggest a novel role for SDCCAG3 in ciliogenesis and in localization of cargo to primary cilia

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules

<p>Abstract</p> <p>Background</p> <p>Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in <it>hydin </it>in <it>hy3 </it>mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown.</p> <p>Results</p> <p>Here we use RNAi in <it>Trypanosoma brucei </it>to address this issue and demonstrate that loss of Hydin causes slow growth and a loss of cell motility. We show that two separate defects in newly-formed flagellar central pair microtubules underlie the loss of cell motility. At early time-points after RNAi induction, the central pair becomes mispositioned, while at later time points the central pair is lost. While the basal body is unaffected, both defects originate at the basal plate, reflecting a role for TbHydin throughout the length of the central pair.</p> <p>Conclusion</p> <p>Our data provide the first evidence of Hydin's role within the trypanosome axoneme, and reveal central pair anomalies and thus impairment of ependymal ciliary motility as the likely cause of the hydrocephalus observed in the <it>hy3 </it>mouse.</p

Congenital Hydrocephalus and Abnormal Subcommissural Organ Development in Sox3 Transgenic Mice

Congenital hydrocephalus (CH) is a life-threatening medical condition in which excessive accumulation of CSF leads to ventricular expansion and increased intracranial pressure. Stenosis (blockage) of the Sylvian aqueduct (Aq; the narrow passageway that connects the third and fourth ventricles) is a common form of CH in humans, although the genetic basis of this condition is unknown. Mouse models of CH indicate that Aq stenosis is associated with abnormal development of the subcommmissural organ (SCO) a small secretory organ located at the dorsal midline of the caudal diencephalon. Glycoproteins secreted by the SCO generate Reissner's fibre (RF), a thread-like structure that descends into the Aq and is thought to maintain its patency. However, despite the importance of SCO function in CSF homeostasis, the genetic program that controls SCO development is poorly understood. Here, we show that the X-linked transcription factor SOX3 is expressed in the murine SCO throughout its development and in the mature organ. Importantly, overexpression of Sox3 in the dorsal diencephalic midline of transgenic mice induces CH via a dose-dependent mechanism. Histological, gene expression and cellular proliferation studies indicate that Sox3 overexpression disrupts the development of the SCO primordium through inhibition of diencephalic roof plate identity without inducing programmed cell death. This study provides further evidence that SCO function is essential for the prevention of hydrocephalus and indicates that overexpression of Sox3 in the dorsal midline alters progenitor cell differentiation in a dose-dependent manner

Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT

Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease