Chemosensory Cilia Research Articles

A single-molecule view on intraflagellar transport in C. elegans chemosensory cilia.

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have been focusing on intraflagellar transport (IFT), which is essential for the assembly and maintenance of cilia. In particular, we study the chemosensory cilia of C. elegans. To visualize IFT with live fluorescence microscopy, we generate mutant worms expressing fluorescent versions of the proteins of interest. Our fluorescence and image-analysis approaches allow us to visualize, track and quantify trains of IFT components moving together, as well as individual motors or IFT proteins. In C. elegans, IFT is driven by groups of tens of three different motor proteins: 2 kinesin-2's (the slow kinesin-II and the faster OSM-3), which drive transport of cargo trains from base to tip of the cilium, and IFT dynein, which drives transport back to the base. Cargo trains move in one go from base to tip and from tip to base. Speed and directionality appear to be regulated by motor proteins docking on and off the trains. Here I present new single-molecule data focusing on the entry of IFT components, including motor proteins, into the cilium. We observe that different IFT components attach to IFT trains in a sequential way, both in time and in location. Pooling the localization information of many individual molecules allows mapping of the 3D ultrastructure of the axoneme with a precision of several tens of nanometers, in wild-type as well as mutant strains lacking e.g. motors. These maps provide new insights in ciliary structure at the base, train-docking dynamics and the interplay of kinesin-II and OSM-3. Our results provide important new insights into how motors cooperate to drive intracellular transport.

Differentiated dynamic response in C.elegans chemosensory cilia.

Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function, cilia are dependent on intraflagellar transport (IFT). Here, we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We find that chemical stimulation results in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents results in different responses, not only in IFT, ciliary structure, and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.

A local interplay between diffusion and intraflagellar transport distributes TRPV-channel OCR-2 along C. elegans chemosensory cilia

To survive, Caenorhabditis elegans depends on sensing soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Cilia rely on intraflagellar transport (IFT) to facilitate the distribution of cargo, such as TPs, along the ciliary axoneme. Here, we use fluorescence imaging of living worms and perform single-molecule tracking experiments to elucidate the dynamics underlying the ciliary distribution of the sensory TP OCR-2. Quantitative analysis reveals that the ciliary distribution of OCR-2 depends on an intricate interplay between transport modes that depends on the specific location in the cilium: in dendrite and transition zone, directed transport is predominant. Along the cilium motion is mostly due to normal diffusion together with a small fraction of directed transport, while at the ciliary tip subdiffusion dominates. These insights in the role of IFT and diffusion in ciliary dynamics contribute to a deeper understanding of ciliary signal transduction and chemosensing.

A single-molecule view on intracellular transport in C. elegans chemosensory cilia

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have focused on a particular transport process, intraflagellar transport (IFT), which takes place in cilia and flagella and is essential for the assembly and maintenance of these organelles. As a model system we use IFT in the chemosensory cilia of the nematode C. elegans. In order to visualize IFT components and cargoes with fluorescence microscopy on living animals, we generate mutant-nematodes expressing fluorescent versions of the proteins of interest.

Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach-diffuse-attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

CDKL kinase regulates the length of the ciliary proximal segment

Cilia are organelles found throughout most unicellular eukaryotes and different metazoan cell types. To accomplish their essential roles in cell motility, fluid flow, and signaling, cilia are divided into subcompartments with variable structures, compositions, and functions. How these specific subcompartments are built remains almost completely unexplored. Here, we show that C.elegans CDKL-1, related to the human CDKL kinase family (CDKL1/CDKL2/CDKL3/CDKL4/CDKL5), specifically controls the length of the proximal segment, a ciliary subdomain conserved in evolution from Tetrahymena motile cilia to C.elegans chemosensory, mammalian olfactory, and photoreceptor non-motile cilia. CDKL-1 associates with intraflagellar transport (IFT), influences the distribution of the IFT anterograde motors heterotrimeric kinesin-II and homodimeric OSM-3-kinesin/KIF17 in the proximal segment, and shifts the boundary between the proximal and distal segments (PS/DS boundary). CDKL-1 appears to function independently from several factors that influence cilium length, namely the kinases DYF-5 (mammalian CILK1/MAK) and NEKL-1 (NEK9), as well as the depolymerizing kinesins KLP-13 (KIF19) and KLP-7 (KIF2). However, a different kinase, DYF-18 (CCRK), is needed for the correct localization and function of CDKL-1 and similarly influences the length of the proximal segment. Loss of CDKL-1, which affects proximal segment length without impairing overall ciliary microtubule structural integrity, also impairs cilium-dependent processes, namely cGMP-signaling-dependent body length control and CO2 avoidance. Collectively, our findings suggest that cilium length is regulated by various pathways and that the IFT-associated kinase CDKL-1 is essential for the construction of a specific ciliary compartment and contributes to development and sensory physiology.

The crowding dynamics of the motor protein kinesin-II.

Intraflagellar transport (IFT) in C. elegans chemosensory cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in large groups to transport cargoes: a fast and processive homodimeric kinesin-2, OSM-3, and a slow and less processive heterotrimeric kinesin-2, kinesin-II. To study the mechanism of the collective dynamics of kinesin-II of C. elegans cilia in an in vitro system, we used Total Internal Reflection Fluorescence microscopy to image the motility of truncated, heterodimeric kinesin-II constructs at high motor densities. Using an analysis technique based on correlation of the fluorescence intensities, we extracted quantitative motor parameters, such as motor density, velocity and average run length, from the image. Our experiments and analyses show that kinesin-II motility parameters are far less affected by (self) crowding than OSM-3. Our observations are supported by numerical calculations based on the TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics). From a comparison of data and modelling of OSM-3 and kinesin-II, a general picture emerges of the collective dynamics of the kinesin motors driving IFT in C. elegans chemosensory cilia and the way the motors deal with crowding.

Single-Molecule Tracking Reveals Complex Motility of Transmembrane Proteins in the Chemosensory Cilia of C. elegans

Cilia are vital for the cell's ability to sense its environment and rely on a process called intra flagellar transport (IFT) for their development, maintenance and function in signal-transduction. In IFT, motor proteins transport ciliary components along the polarized microtubule axoneme of the cilium. Among the cargoes of IFT are transmembrane proteins involved in signal transduction. As a model system, we study C. elegans chemosensory cilia, which we study using fluorescence microscopy with single-molecule sensitivity. First, we demonstrate that IFT machinery and ciliary components, including TRPV transmembrane channel protein OCR-2 are redistributed away from the ciliary tip upon external chemical stimulation, in a robust, extensive and reversible way. To elucidate the dynamics underlying this dramatic protein redistribution, we performed single-molecule imaging of OCR-2 in live C. elegans. Advanced analysis of the single-molecule trajectories shows that, in dendrite and transition zone, active transport is the prevailing motility mode of OCR-2. In the proximal and distal segments, however, motility is a much more complex, location-specific interplay between active transport, normal diffusion and sub diffusion. At the tip, confinement of the membrane proteins plays an important role. Together, our data and analysis demonstrate an intricate interplay between modes of transportation that ensure the proper ciliary distribution of OCR-2. These insights in the dynamics of cellular signal-transduction contributes to a wider understanding of IFT dynamics and to cilia as chemosensory organelles.

A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl\u2212 current when transfected into HEK293

BackgroundCLCA is a family of metalloproteases that regulate Ca2+-activated Cl− fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl− current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl− channel. We explored the possibility that this protein plays a role in odor transduction.ResultsWe cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl−-selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl− current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2.ConclusionThese findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl− channel involved in odor transduction.

Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose.

The mechanisms that power the physiological events occurring in cilia, flagella, and microvilli are of fundamental importance for the functions of these important and ubicuous organelles. The olfactory epithelium is mostly populated by ciliated olfactory sensory neurons (OSNs) and surrounding sustentacular cells (SCs) with apical microvilli. The only OSN dendrite extends to the surface forming a knob projecting several chemosensory cilia of ∼50 × 0.2 μm, devoid of inner membranes embedded in a mucus layer. Upon odorant binding, odor receptors couple to G-protein activating adenylyl cyclase, producing cAMP. cAMP opens cyclic nucleotide-gated channels allowing a Ca2+ influx that opens Ca2+-activated Cl- channels, generating the receptor potential. Many enzymes are activated in chemotransduction to hydrolyze ATP. The knob contains approximately two mitochondria; assuming that the cilia ATP is 1 mm and diffuses along it at ∼10 μm in 500 ms, ATP from the knob mitochondria may not fulfill the demands of transduction over the full length of the cilium, which suggests an additional ATP source. We measured millimolar glucose in rat mucus; we detected glucose transporter GLUT3 in rat and toad (Caudiverbera caudiverbera) OSN cilia, SC microvilli, and glycolytic enzymes in rat cilia. We also found that the cilia and knob can incorporate and accumulate 2-deoxyglucose (glucose analog), but not when blocking GLUT. Glucose removal and the inhibition of glycolysis or oxidative phospholylation impaired the odor response. This evidence strongly suggests that glycolysis in the cilia and knob oxidative phosphorylation together fuel chemotransduction.SIGNIFICANCE STATEMENT How processes occurring in cilia and flagella are powered is a matter of general interest. Substantial progress has been made in unraveling the sensory transduction mechanisms, commonly occurring in such structures; however, the energy sources powering them have been scarcely explored. Accessibility to the specialized sensory organelles and their small dimensions have been limiting factors. Olfactory sensory neurons chemosensory cilia are elongated, mucus embedded, fully exposed structures particularly amenable for a multidisciplinary study of this problem, as done here. We demonstrate the occurrence and functionality of glucose uptake and glycolysis in the cilia. We support that odor transduction relies on ATP generated by oxidative phosphorylation in the dendrite and glycolytically in the cilia using glucose internalized from the mucus.

Single Ca(2+)-activated Cl(-) channel currents recorded from toad olfactory cilia.

BackgroundOdor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca2+-activated Cl− channels, allowing a Cl− inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca2+-activated Cl− channel of olfactory transduction. Although Ca2+-activated Cl− channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca2+-activated Cl− channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca2+-activated Cl− channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved.ResultsWe explored the presence of detectable Ca2+-activated Cl− channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl− current events with a pronounced Ca2+ dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca2+ affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction.ConclusionsThese results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

Why motor proteins team up - Intraflagellar transport inC. eleganscilia

ABSTRACTInside the cell, vital processes such as cell division and intracellular transport are driven by the concerted action of different molecular motor proteins. In C. elegans chemosensory cilia, 2 kinesin-2 family motor proteins, kinesin-II and OSM-3, team up to drive intraflagellar transport (IFT) in the anterograde direction, from base to tip, whereas IFT dynein hitchhikes toward the tip and subsequently drives IFT in the opposite, retrograde direction, thereby recycling both kinesins. While it is evident that at least a retrograde and an anterograde motor are necessary to drive IFT, it has remained puzzling why 2 same-polarity kinesins are employed. Recently, we addressed this question by combining advanced genome-engineering tools with ultrasensitive, quantitative fluorescence microscopy to study IFT with single-molecule sensitivity.1,2 Using this combination of approaches, we uncovered a differentiation in kinesin-2 function, in which the slower kinesin-II operates as an ‘importer’, loading IFT trains into the cilium before gradually handing them over to the faster OSM-3. OSM-3 subsequently acts as a long-range ‘transporter’, driving the IFT trains toward the tip. The two kinesin-2 motors combine their unique motility properties to achieve something neither motor can achieve on its own; that is to optimize the amount of cargo inside the cilium. In this commentary, we provide detailed insight into the rationale behind our research approach and comment on our recent findings. Moreover, we discuss the role of IFT dynein and provide an outlook on future studies.

Properties of an optogenetic model for olfactory stimulation.

In olfactory research it is difficult to deliver stimuli with defined intensity and duration to olfactory sensory neurons. Expression of channelrhodopsin 2 (ChR2) in olfactory sensory neurons provides a means to activate these neurons with light flashes. Appropriate mouse models are available. The present study explores the suitability of an established olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) mouse model for ex vivo experimentation. Expression of ChR2 in sensory neurons of the main olfactory epithelium, the septal organ and vomeronasal organ is characterized. Expression pattern of ChR2 in olfactory receptor neurons and the properties of light responses indicate that light stimulation does not impact on signal transduction in the chemosensory cilia. Light-induced electro-olfactograms are characterized with light flashes of different intensities, durations and frequencies. The impact of light-induced afferent stimulation on the olfactory bulb is examined with respect to response amplitude, polarity and low-pass filtering. For the examination of sensory processing, it is helpful to deliver stimuli in precisely defined temporal and spatial patterns with accurate control of stimulus intensity. This is challenging in experiments with the mammalian olfactory system because airborne odorants have to be transported into the intricate sensory structures of the nose and must dissolve in mucus to be detected by sensory neurons. Defined and reproducible activity can be generated in olfactory sensory neurons that express the light-gated ion channel channelrhodopsin 2 (ChR2). The neurons can be stimulated by light flashes in a controlled fashion by this optogenetic approach. Here we examined the application of an olfactory marker protein (OMP)/ChR2-yellow fluorescent protein (YFP) model for ex vivo exploration of the olfactory epithelium and the olfactory bulb of the mouse. We studied the expression patterns of ChR2 in the main olfactory system, the vomeronasal system, and the septal organ, and we found that ChR2 is absent from the sensory cilia of olfactory sensory neurons. In the olfactory epithelium, we characterized light-induced electro-olfactograms with respect to peripheral encoding of stimulus intensity, stimulus duration and stimulus frequency. In acute slices of the olfactory bulb, we identified specific aspects of the ChR2-induced input signal, concerning its dynamic range, its low-pass filter property and its response to prolonged stimulation. Our study describes the performance of the OMP/ChR2-YFP model for ex vivo experimentation on the peripheral olfactory system and documents its versatility and its limitations for olfactory research.

Dynein Dynamics in Vivo: A Single-Molecule View on its Function in Intraflagellar Transport

IFT-dynein (cytoplasmic dynein 2) co-operates with kinesin-2 motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). Relatively little is known about this dynein, while the related cytoplasmic dynein 1 has received a lot of attention of late. Here, we study the in vivo IFT-dynein dynamics at the ensemble and single-molecule level.To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors, expressed at endogenous levels, in the chemosensory cilia of living C. elegans. Fluorescence movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using custom kymograph-analysis software. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, to track individual IFT-dynein motors in vivo.The data revealed that IFT-dynein moves in trains consisting tens of motor proteins. Retrograde trains are smaller but more frequent than anterograde (kinesin-driven) trains. Anterograde and retrograde IFT-dynein flux are equal along cilia, indicating that the cilium is a closed system for dynein. While the kinesin composition of a train varies along the track, the amount of dynein per train remains relatively constant. Remarkably, this does not result in directionality changes along the track, like in reported ‘tug-of-war’ motor systems, suggesting that retrograde and anterograde motor activities are carefully orchestrated in IFT.Single IFT-dynein measurements support the ensemble findings. Trajectories show distinct motility behavior: diffusion at the ciliary base, pauses, turns and directed motion. Pauses in retrograde or anterograde trajectories are never followed by a directional switch. Moreover, retrograde-to-anterograde turn events are rare.This combined ensemble and single-molecule approach has provided new insights into IFT-dynein transport dynamics in living organisms, shedding light on dynein function in general.

Quantification of IFT-Dynein Dynamics in C.elegans

Cytoplasmic dyneins are the main drivers of microtubule-based retrograde transport in eukaryotic cells. Cytoplasmic dynein 1 plays a role in retrograde intracellular transport and cell division, whereas cytoplasmic dynein 2, also known as IFT-dynein, co-operates with kinesin motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). While cytoplasmic dynein 1 has been the subject of many recent studies, relatively little is known about IFT-dynein. Here, we focus on the mechanism and dynamics of IFT-dynein: how does it behave in vivo at the ensemble and single-molecule level?To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors in the chemosensory cilia of living C. elegans. Transgene worms were generated using the Mos1-mediated single copy insertion (MosSCI) method to ensure endogenous motor expression levels. Time-lapse fluorescence movies showed that IFT-dynein moves in trains consisting of tens of motor proteins. The movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using in-house developed kymograph-analysis software. This analysis revealed that IFT-dynein train velocities and motor numbers are dynamic, changing along the cilium. Double-labeled constructs allowed us to look more closely into motor co-operation, determining the dynein:kinesin ratio at different positions in the cilium.To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, the tracking of individual IFT-dynein motors in vivo. Single-motor trajectories revealed distinct features of IFT-dynein motility: diffusive behavior at the ciliary base, pauses, turns, directed motion and switches between these behaviors. This combined ensemble and single-molecule approach has provided novel quantitative insight into IFT-dynein dynamics in living organisms, shedding light on the complex functioning of dynein motors in general.

A Single-Molecule View on Kinesin Motor-Protein Cooperation in Intraflagellar Transport in Living C. elegans

Intraflagellar transport (IFT) is an essential intracellular transport mechanism in cilia, the hair-like, microtubule-based protrusions of eukaryotic cells with sensory or motile functions. In the chemosensory cilia of the nematode C. elegans, IFT is driven by the cooperative action of IFT-dynein (responsible for transport from cilium tip to base) and two kinesin motor proteins, Kinesin-II and OSM-3, (responsible for transport in the opposite direction). Our goal was to understand why two kinesins are needed for IFT and what their respective roles are. To achieve this, we generated mutant nematodes expressing fluorescent versions of the motor proteins at endogenous levels and subjected them to in vivo fluorescence microscopy with single-molecule resolution. Images obtained were analyzed using automated kymograph and single-particle tracking analysis, providing unprecedented, quantitative insight in the role of the kinesins in IFT. We find that the two kinesins fulfill distinct roles in line with their distinct motility properties. Kinesin-II is the slower and less processive motor. In IFT its key role is to load of IFT trains, to initiate the transport of multiple, coupled motor proteins connected to cargo, and to effectively traverse the transition zone, the semi-permeable protein barrier between cilium and rest of the cell. After successful crossing of the transition zone, Kinesin-II leaves the trains and the other, faster and more processive kinesin, OSM-3 binds and drives the longer-distance transport to the cilium tip. Our results provide insight in how cells use a combination of motor proteins to drive intracellular transport and demonstrate the power of single-molecule fluorescence microscopy to unravel complex processes in the cells of living, multicellular organisms.

The Extracellular Matrix Protein Artichoke Is Required for Integrity of Ciliated Mechanosensory and Chemosensory Organs inDrosophilaEmbryos

Sensory cilia are often encapsulated by an extracellular matrix (ECM). In Caenorhabditis elegans, Drosophila melanogaster, and vertebrates, this ECM is thought to be directly involved in ciliary mechanosensing by coupling external forces to the ciliary membrane. Drosophila mechano- and chemosensory cilia are both associated with an ECM, indicating that the ECM may have additional roles that go beyond mechanosensory cilium function. Here, we identify Artichoke (ATK), an evolutionarily conserved leucine-rich repeat ECM protein that is required for normal morphogenesis and function of ciliated sensilla in Drosophila. atk is transiently expressed in accessory cells in all ciliated sensory organs during their late embryonic development. Antibody stainings show ATK protein in the ECM that surrounds sensory cilia. Loss of ATK protein in atk null mutants leads to cilium deformation and disorientation in chordotonal organs, apparently without uncoupling the cilia from the ECM, and consequently to locomotion defects. Moreover, impaired chemotaxis in atk mutant larvae suggests that, based on ATK protein localization, the ECM is also crucial for the correct assembly of chemosensory receptors. In addition to defining a novel ECM component, our findings show the importance of ECM integrity for the proper morphogenesis of ciliated organs in different sensory modalities.

Roles of dopamine receptor on chemosensory and mechanosensory primary cilia in renal epithelial cells

Dopamine plays a number of important physiological roles. However, activation of dopamine receptor type-5 (DR5) and its effect in renal epithelial cells have not been studied. Here, we show for the first time that DR5 is localized to primary cilia of LLCPK kidney cells. Renal epithelial cilia are mechanosensory organelles that sense and respond to tubular fluid-flow in the kidney. To determine the roles of DR5 and sensory cilia, we used dopamine to non-selectively and fenoldopam to selectively activate ciliary DR5. Compared to mock treatment, dopamine treated cells significantly increases the length of cilia. Fenoldopam further increases the length of cilia compared to dopamine treated cells. The increase in cilia length also increases the sensitivity of the cells in response to fluid-shear stress. The graded responses to dopamine- and fenoldopam-induced increase in cilia length further show that sensitivity to fluid-shear stress correlates to the length of cilia. Together, our studies suggest for the first time that dopamine or fenoldopam is an exciting agent that enhances structure and function of primary cilia. We further propose that dopaminergic agents can be used in “cilio-therapy” to treat diseases associated with abnormal cilia structure and/or function.

Illuminating the Cooperative Action of Kinesin-II and OSM-3-Kinesin in the Chemosensory Cilia of Caenorhabditis Elegans

Intraflagellar transport (IFT) is indispensable for the assembly and maintenance of cilia. In the chemosensory cilia of C. elegans, two motors of the kinesin-2-family, heterotrimeric kinesin-II and homodimeric OSM-3, collaborate to drive anterograde IFT. It is known that both kinesins associate with IFT-trains that move along the middle segment, and that OSM-3 alone drives transport in the distal segment. However, many questions remain concerning how these kinesins cooperate, how this is regulated and why both motors are required. To address this problem, we have improved the fidelity of IFT-assays by using single transgenes encoding for fluorescently-labeled kinesins. In combination with ultrasensitive, quantitative fluorescence microscopy and dynamic photoactivated localization microscopy allowing observation of single motors in living nematodes, we find that the motor composition of IFT-trains is highly dynamic. Kinesin-II undocks gradually from IFT-trains close to the ciliary base, and not suddenly upon reaching the middle segment tips as previously thought, whereas OSM-3 docks gradually resulting in accelerating IFT-trains. Undocked kinesin-II is transported back to the ciliary base by dynein-driven retrograde trains. Consequently, the IFT-system ensures that kinesin-II stays close to the ciliary base, where, it is responsible for the loading of IFT-trains onto the axoneme, and that OSM-3 stays around the distal segment, where it drives fast long-distance transport. Our work on motor cooperation and dynamics provides new insight into how IFT drives the proper development and functioning of cilia and, more broadly, how kinesin motors work together to generate intracellular transport pathways within cells.

Development of an integrated microfluidic device for evaluating of in vivo chemo-sensing of intact Caenorhabditis elegans

Conventional macro-scale methods are not appropriate for in vivo neuronal analysis at a single neuron level when experimental subject is small model organism Caenorhabditis elegans. In this paper, we demonstrate a powerful microfluidic device that allowed investigating in vivo chemo-sensing of intact C. elegans by integrating functions of automated manipulation of worms, effective immobilization without damages and well-controllable stimulations. Live C. elegans were effectively immobilized in microfluidic channels by a comb-shaped microvalve without physiological damages. A well-controlled sample introduction system was used to deliver chemicals to the chemosensory cilia at the worm's nose tip. In vivo neuronal responses of the animal to the chemical stimulations were monitored using calcium fluorescence imaging. Dynamic calcium signals related to varying concentrations and durations of stimuli were investigated using this integrated microfluidic device. We anticipate this immobilization approaches can have an impact in a variety of applications, such as neuron responses investigation before and after laser ablation upstream or downstream neurons for studying olfactory or gustatory neural circuits in individual healthy worms, in this high-throughput system.