Ciliary Transport Research Articles

Common general anesthetic propofol impairs kinesin processivity

Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABAA receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.

IFT81 as a Candidate Gene for Nonsyndromic Retinal Degeneration.

PurposeIFT81, a core component of the IFT-B complex, involved in the bidirectional transport of ciliary proteins, has been recently implicated in syndromic ciliopathies. However, none of the IFT-B core complex proteins have been associated with nonsyndromic retinal dystrophies. Given the importance of ciliary transport in photoreceptor function and structural maintenance, we sought to investigate the impact of IFT (intraflagellar transport) mutations in nonsyndromic retinopathies.MethodsWhole exome sequencing was performed on 50 cone-rod dystrophy (CRD) patients that were previously screened for mutations in known retinal disease genes. The impact of candidate mutation was studied using in vitro cell system and in vivo zebrafish assay to determine the pathogenicity of the variant.ResultsCompound heterozygous mutations in IFT81, including one nonsense (c.1213C>T, p.R405*) and one missense variant (c.1841T>C, p.L614P), were identified in a nonsyndromic CRD proband. Extensive functional analyses of the missense variant in cell culture and zebrafish strongly suggests its pathogenic nature. Loss of IFT81 impairs ciliogenesis and, interestingly, the missense variant displayed significantly reduced rescue of ciliogenesis in the IFT81 knockdown in vitro system. Consistently, dramatic reduction of rescue efficiency of the ift81 mutant zebrafish embryo by mRNA with the missense variant was observed, further supporting its pathogenicity.ConclusionsConsistent with the function of the IFT-B complex in the maintenance of photoreceptor cilium, we report a case of mutations in a core IFT-B protein, IFT81. This represents the first report of mutations in IFT81 as a candidate gene for nonsyndromic retinal dystrophy, hence expanding the phenotype spectrum of IFT-B components.

Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration.

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4’s role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

Cell scientist to watch – Gaia Pigino

ABSTRACT Gaia Pigino received her master's degree in natural science followed by a PhD in evolutionary biology – focusing on zoology and ecotoxicology – in the laboratory of Fabio Bernini at the University of Siena, Italy. She then joined the electron microscopy laboratory of Pietro Lupetti for her first postdoctoral position before moving to Zürich. There, supported by an EMBO Long-Term Fellowship, she worked with Takashi Ishikawa at the Swiss Federal Institute of Technology and the Paul Scherrer Institute in Villigen. In 2012, Gaia became an independent group leader at the Max Planck Institute in Dresden. Her laboratory is interested in understanding the assembly of the cilium and mechanisms of ciliary transport in eukaryotic cells, as investigated with cryo-transmission electron microscopy.

Intraflagellar Transport and Ciliary Dynamics.

Cilia and flagella are microtubule-based organelles whose assembly requires a motile process, known as intraflagellar transport (IFT), to bring tubulin and other components to the distal tip of the growing structure. The IFT system uses a multiprotein complex with components that appear to be specialized for the transport of different sets of cargo proteins. The mechanisms by which cargo is selected for ciliary import and transport by IFT remain an area of active research. The complex dynamics of cilia and flagella are under constant regulation to ensure proper length control, and this regulation appears to involve regulation at the stage of IFT injection into the flagellum, as well as regulation of flagellar disassembly and, possibly, of cargo binding. Cilia and flagella thus represent a convenient model system to study how multiple motile and signaling pathways cooperate to control the assembly and dynamics of a complex cellular structure.

Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling.SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function.

The Intraflagellar Transport Machinery.

Eukaryotic cilia and flagella are evolutionarily conserved organelles that protrude from the cell surface. The unique location and properties of cilia allow them to function in vital processes such as motility and signaling. Ciliary assembly and maintenance rely on intraflagellar transport (IFT), the bidirectional movement of a multicomponent transport system between the ciliary base and tip. Since its initial discovery more than two decades ago, considerable effort has been invested in dissecting the molecular mechanisms of IFT in a variety of model organisms. Importantly, IFT was shown to be essential for mammalian development, and defects in this process cause a number of human pathologies known as ciliopathies. Here, we review current knowledge of IFT with a particular emphasis on the IFT machinery and specific mechanisms of ciliary cargo recognition and transport.

Female rat transcriptome response to infraorbital nerve transection differs from that of males: RNA-seq.

The effects of infraorbital nerve (ION) transection on gene expression in the adult female rat barrel cortex were investigated using RNA sequencing. After a 24-hour survival duration, 28 genes were differentially regulated by ION transection. Differentially expressed genes suggest microglial activity, increased retrograde ciliary transport, and a decrease in inhibition. These changes may be functionally comparable to changes in the male barrel cortex, where changes in genes related to morphology, neuronal activity, and neuronal excitability were observed. However, the patterns in changes in gene expression are vastly different between male and female rats. The results strongly caution against the practice of generalizing data from one sex to both sexes. This cautionary note has potentially profound implications for a range of research lines, including substance abuse and stress, both research domains in which subjects have been predominantly males. Future research needs to employ sex as a classification variable, as sex differences can generally be expected. Future research is also needed to confirm that changes in gene expression observed with RNA-seq correlate with changes in protein expression. J. Comp. Neurol. 525:140-150, 2017. © 2016 Wiley Periodicals, Inc.

WS03.2 MKA 104, a long-acting ENaC down-regulator, enhances speed of ciliary transport of secretions on wild-type and CFTR(–/–) pig trachea with greater efficacy and duration than existing CF treatments

WS03.2 MKA 104, a long-acting ENaC down-regulator, enhances speed of ciliary transport of secretions on wild-type and CFTR(–/–) pig trachea with greater efficacy and duration than existing CF treatments

Autosomal recessive IFT57 hypomorphic mutation cause ciliary transport defect in unclassified oral-facial-digital syndrome with short stature and brachymesophalangia.

The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.

PDE6δ-mediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity.

The phosphodiesterase 6 delta subunit (PDE6δ) shuttles several farnesylated cargos between membranes. The cargo sorting mechanism between cilia and other compartments is not understood. Here we show using the inositol polyphosphate 5′-phosphatase E (INPP5E) and the GTP-binding protein (Rheb) that cargo sorting depends on the affinity towards PDE6δ and the specificity of cargo release. High-affinity cargo is exclusively released by the ciliary transport regulator Arl3, while low-affinity cargo is released by Arl3 and its non-ciliary homologue Arl2. Structures of PDE6δ/cargo complexes reveal the molecular basis of the sorting signal which depends on the residues at the −1 and −3 positions relative to farnesylated cysteine. Structure-guided mutation allows the generation of a low-affinity INPP5E mutant which loses exclusive ciliary localization. We postulate that the affinity to PDE6δ and the release by Arl2/3 in addition to a retention signal are the determinants for cargo sorting and enrichment at its destination.

Novel small molecules targeting ciliary transport of Smoothened and oncogenic Hedgehog pathway activation.

Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics.

Methods for Studying Movement of Molecules Within Cilia.

The assembly of cilia and eukaryotic flagella (interchangeable terms) requires the import of numerous proteins from the cell body into the growing organelle. Proteins move into and inside cilia by diffusion and by motor-based intraflagellar transport (IFT). Many aspects of ciliary protein transport such as the distribution of unloading sites and the frequency of transport can be analyzed using direct in vivo imaging of fluorescently tagged proteins. Here, we will describe how to use total internal reflection fluorescence microcopy (TIRFM) to analyze protein transport in the flagella of the unicellular alga Chlamydomonas reinhardtii, a widely used model for cilia and cilia-related disease.

Hedgehog Signaling Regulates the Ciliary Transport of Odorant Receptors in Drosophila.

Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. Here, we show that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). We demonstrate that this is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). We further demonstrate that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand our knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking.

A Group of ent-Kaurane Diterpenoids Inhibit Hedgehog Signaling and Induce Cilia Elongation.

The Hedgehog (Hh) signaling pathway plays important roles in the tumorigenesis of multiple cancers and is a key target for drug discovery. In a screen of natural products extracted from Chinese herbs, we identified eight ent-Kaurane diterpenoids and two triterpene dilactones as novel Hh pathway antagonists. Epistatic analyses suggest that these compounds likely act at the level or downstream of Smoothened (Smo) and upstream of Suppressor of Fused (Sufu). The ent-Kauranoid-treated cells showed elongated cilia, suppressed Smo trafficking to cilia, and mitotic defects, while the triterpene dilactones had no effect on the cilia and ciliary Smo. These ent-Kaurane diterpenoids provide new prototypes of Hh inhibitors, and are valuable probes for deciphering the mechanisms of Smo ciliary transport and ciliogenesis.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation.

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Modulation of Ciliary Phosphoinositide Content Regulates Trafficking and Sonic Hedgehog Signaling Output

Ciliary transport is required for ciliogenesis, signal transduction, and trafficking of receptors to the primary cilium. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) have been associated with ciliary dysfunction; however, its role in regulating ciliary phosphoinositides is unknown. Here we reportthat in neural stem cells, phosphatidylinositol 4-phosphate (PI4P) is found in high levels in cilia whereas phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is not detectable. Upon INPP5E inactivation, PI(4,5)P2 accumulates at the ciliary tip whereas PI4P is depleted. This is accompanied by recruitment of the PI(4,5)P2-interacting protein TULP3 to the ciliary membrane, along with Gpr161. This results in an increased production of cAMP and a repression of the Shh transcription gene Gli1. Our results reveal the link between ciliary regulation of phosphoinositides by INPP5E and Shh regulation via ciliary trafficking of TULP3/Gpr161 and also provide mechanistic insight into ciliary alterations found in Joubert and MORM syndromes resulting from INPP5E mutations.

Rab23 has a novel role in the trafficking of Kif17 to the primary cilia

Rab GTPases are regulators of the intracellular vesicular transport and interorganelle protein trafficking of both endocytic and secretory pathways. The small GTPase Rab23 is an antagonist of the Sonic hedgehog (Shh) signaling pathway during mouse embryonic development, but its exact role and mode of mechanism have remained elusive. Since modulation of Shh signaling depends on normal functioning of primary cilia, and Evi5L's overexpression (Rab23's RabGAP) led to decreases in primary ciliogenesis, Rab23 likely has a role at the cilium. We found Rab23 wild-type and constitutively active Rab23Q68L mutant enriched at the primary cilia. In testing the effect of Rab23's manipulations on the ciliary localization of several known ciliary cargoes, ciliary localization of a kinesin-II motor protein Kif17 turned out to be disrupted by overexpression of dominant negative Rab23S23N and in Rab23 depleted cells. In addition, Kif17's ciliary mislocalization could be rescued after expression of a non-degradable wild-type Rab23 and FRAP experiments showed that Rab23 silenced cells exhibited reduced recovery of ciliary Kif17 after photobleaching. Co-immunoprecipitation studies further revealed that Rab23 exists in a tripartite complex with Kif17 and Importin β2 (Kif17's ciliary import carrier), implying that Kif17 requires binding to regulatory proteins like Rab23 for its ciliary transport. Although a ciliary-cytoplasmic gradient of nuclear Ran is necessary in regulating Kif17's ciliary import, both small GTPases Rab23 and Ran appear to have independent roles in the ciliary entry of Kif17. Our findings have uncovered a hitherto unknown effector of Rab23 and demonstrated how Rab23 could mediate Kif17's trafficking to the primary cilium.

Transgenic tools for proteomic analysis of ciliary transport

Vision begins as photons are captured by photoreceptor cilia and light is converted into electrical signals that are then sent to the brain. As the photoreceptor cilium is not able to make its own proteins, all polypeptides needed for converting photons into electrical signals are synthetized in the cell body. How these molecules move from the cell body to cilia is still unclear. Opsin is one of the best-characterized transmembrane proteins. Our goal is to understand the mechanism of opsin transport into photoreceptor cilia. In this project, we use a combination of genetic and proteomic approaches in the zebrafish model. As the first step, we are constructing a transgenic line that expresses EGFP-opsin fusion from an inducible promoter specifically in photoreceptors. For this purpose, we will mate two transgenic lines: 1. A line that specifically expresses Cre recombinase in photoreceptors, and 2. A line that conditionally expresses EGFP-opsinCT44 (EGFP fused with the 44 C-terminal residues of opsin, which are sufficient to mediate ciliary transport) from the heat-shock promoter. A lox-mCherry-STOP cassette is inserted upstream of EGFP-opsinCT44. In the long run, we plan to purify photoreceptors via FACS sorting, pull down EGFP-opsinCT44 using the EGFP tag, and analyze opsin C-terminal binding proteins by mass spectrometry. In parallel, we will perform analysis of opsin transport in cilia mutants. We hope this will allow us to formulate a general model of how transmembrane proteins, such as GPCRs and TRP channels, are transported into cilia.

Identification of C. elegans RAB-5-dependent downstream effectors as regulators of ciliogenesis and ciliary membrane homeostasis

Cilia are microtubule based organelles enveloped by a specialised extension of plasma membrane. Serving as cellular antennae, cilia are essential for perceiving extracellular cues and mediating downstream signal transduction crucial for normal cellular physiology and development. These functions are mediated in large part by the specialised ciliary membrane, which differs in composition from the plasma membrane, housing many of the receptors, channels and effectors that mediate cilium-based signalling. However, the molecular mechanisms underlying the establishment, maintenance and regulation of the ciliary membrane remain poorly understood. In this project we are seeking to identify and functionally characterize genes with ciliogenic roles in ciliary membrane transport and organisation. Specifically, we are taking a candidate gene approach using the Caenorhabditis elegans ciliated sensory neuronal model, employing assays for cilium integrity (dye filling assay) and functionality (osmotic avoidance assay; foraging). Using predicted loss-of-function alleles, we screened 55 mutants of evolutionarily conserved membrane organising and trafficking genes and identified 4 rab-5-related endocytic gene mutants with defects in cilium integrity and function. One of these, rabs-5, encodes a RAB-5 effector, which we found is expressed mainly in ciliated cells. rabs-5 mutants possess short cilia and an expanded periciliary membrane compartment, indicative of defects in ciliary membrane homeostasis. Transgenic expression of a rabs-5(WT) construct rescues the cilium integrity and foraging defects of rabs-5 mutants, confirming the association of rabs-5 with cilia-related functions. By taking a reverse genetics candidate gene approach in C. elegans, a number of RAB-5-dependent downstream effectors have been identified as potential regulators of ciliogenesis and ciliary membrane homeostasis.