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Soluble levels of cytosolic tubulin regulate ciliary length control

Recent Activity. Affiliations 1. No matching affiliation detected. Find all citations in this journal default. Or filter your current search. Methods in Cell Biology [23 Dec , ]. Type: Research Support, Non-U. Abstract The Hedgehog Hh signal transduction pathway is essential for the development and patterning of numerous organ systems, and has important roles in a variety of human cancers.

Genetic screens for mouse embryonic patterning mutants first showed a connection between mammalian Hh signaling and intraflagellar transport IFT , a process required for construction of the primary cilium, a small cellular projection found on most vertebrate cells. Additional genetic and cell biological studies have provided very strong evidence that mammalian Hh signaling depends on the primary cilium. Here, we review the evidence that defines the integral roles that IFT proteins and cilia play in the regulation of the Hh signal transduction pathway in vertebrates.

We discuss the mechanisms that control localization of Hh pathway proteins to the cilium, focusing on the transmembrane protein Smoothened Smo , which moves into the cilium in response to Hh ligand. The phenotypes caused by loss of cilia-associated proteins are complex, which suggests that cilia and IFT play active roles in mediating Hh signaling rather than serving simply as a compartment in which pathway components are concentrated.

Hh signaling in Drosophila does not depend on cilia, but there appear to be ancient links between cilia and components of the Hh pathway that may reveal how this fundamental difference between the Drosophila and mammalian Hh pathways arose in evolution. The current—voltage relation was measured in each of two pseudointracellular baths: a bath containing 0.

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If the pipette was raised briefly into the air, the cell was pulled off at the air-bath interface, while the cilium remained sealed inside the pipette in the inside-out recording configuration. The pipette and cilium were then quickly transferred through the air and immersed in a solution that bathed the cytoplasmic face of the membrane. Such transfers could be repeated many times without rupturing the seal. Initially each cilium was placed in a pseudointracellular bath containing 0. The current—voltage relation measured in this bath was linear Figure 3 B , and the input resistance averaged Clear single-channel events could not be discriminated, suggesting that the current reflects the summated activities of a moderate number of smaller-conductance channels.

Our goal was to allow the direct study of electrical signals in the membrane of a primary cilium. To develop a method for this, we selected the cilia of cultured renal epithelial cells for two reasons.

The primary cilium

First, as discussed below, an existing body of work has identified specific channels and receptors that are speculated to underlie sensory functions in renal cilia. Second, channel defects in renal cilia are implicated in cystic kidney diseases, including autosomal dominant polycystic kidney disease ADPKD [ 6 ], the most common monogenic renal disorder [ 44 ]. In pilot studies, we were unable to record from the cilia of cells attached to fixed substrates such as cover glasses or folded filter paper [ 48 ]. On cover glasses, visual identification of the cilia was difficult because of the underlying cells.

Primary Cilia, Volume 94 - 1st Edition

Even on folded filter paper, the base of the cilium could not be clearly seen. As a result, we were unable to move the recording pipette along the full length of a cilium to its base, where the pipette can seal to the membrane. This problem was overcome by growing the cells on a movable substrate, a glass-coated bead small enough to be easily moved by suction applied through the recording pipette. The full length of a cilium was able to follow this suction until the pipette sealed near the base of the cilium. Obtaining such seals was also possible with enzymatically dissociated cells, but that introduces a risk of enzymatic degradation of membrane proteins.

It is of central importance to know that the primary cilium enters the recording pipette. This was evident from direct observation Figure 2 Additional file 1. The initial recording studies also suggested the presence of a specialized membrane. We identified a large-conductance channel Figure 3 B,C that was present in ciliary recordings but not in patches from the apical cell membrane.

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Although this method allows a focus on the ciliary membrane, it is likely that some plasma membrane surrounding the base of the cilium also enters the pipette [ 49 ]. Two limitations apply to our method at present.

First, the cilium inside the pipette cannot be bent with fluid flow, which limits the ability to study mechanical stimulation. In the future, an appropriate ciliary marker might allow visualization of even a short cilium within the recording pipette following recording. The first direct recordings from the membrane of a primary cilium were reported by Raychowdhury et al.

Primary cilia were isolated from cultured cells derived from porcine renal epithelium, and membrane patches were excised from the cilia. Our method offers three improvements. First, we record from each cilium while it is cell-attached or immediately after it is plucked from the cell.

In the previous method [ 16 ], isolating the cilia by centrifugation took over two hours. The cilia were then frozen and thawed for subsequent use. Second, recording from small excised patches [ 16 ] samples the membrane and reduces the chances of observing channels expressed at low density or at specific loci on the cilia.

Primary Cilia, Volume 94 (Methods in Cell Biology)

We record from the entire membrane of one cilium. Finally, the authors of the published method noted that low input resistances constrained their analyses [ 16 ]. As indicated above, we achieve very high input resistances. The growth of ciliated cells on beads may prove useful in applications other than electrophysiological recording.

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Proteins have been isolated and identified from single cells, but the sensitivity is not high [ 51 ]. It is conceivable, particularly with longer cilia and future improvements in proteomic techniques, that micropipettes could be used to isolate enough cilia from cell-coated beads to generate a primary ciliome. An alternative approach would be to culture cells on beads in a spinner flask so that a large number of ciliated cells would be grown in a small space. Subsequent isolation of the cilia by established means [ 52 ] might suffice for the purpose of making a primary ciliome.

The method for recording from primary cilia was developed with cultured renal epithelial cells. We must acknowledge that cultured cells cannot perfectly represent the environment that exists in the inner medullary collecting duct of the murine kidney. There is evidence that protein localization to primary cilia is altered by environmental factors such as flow [ 53 ], availability of ligands [ 54 ], and serum deprivation [ 55 ].

However, culture conditions similar to ours have been successfully used by other researchers studying ciliary function in kidney cells [ 28 , 56 ].

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  7. In the future, it may be productive to use acutely dissociated preparations from patient samples despite the inherent risks of enzymatic damage to membrane proteins. A technique that measures ciliary electrical activity will be useful in examining ciliary signaling pathways that use ion channels. It seems likely that different cells will have different channels localized to their primary cilia for the transduction of different stimuli.

    For example, TRPP2 is present on the primary cilium of the following cells and has been hypothesized or shown to transduce mechanical stimuli: embryonic node cells, ovarian granulosa cells, cholangiocytes, vascular smooth muscle cells, and vascular endothelial cells [ 59 , 60 , 61 , 62 , 63 ]. TRPV4 is present on the primary cilia of cholangiocytes and mediates a response to changes in osmolarity [ 8 ].

    In neurons, several receptors for channel-containing pathways have been localized to primary cilia for example, melanin-concentrating hormone receptor 1 [ 64 , 65 ] , although corresponding ciliary channels have not yet been found. The utility of our method should increase when coupled with methods for manipulating the expression of ciliary proteins. Targeted ciliary expression has been possible in IMCD [ 54 , 66 ] and other cell lines [ 67 , 68 , 69 ].

    Recording from cells treated to knock down ciliary channel proteins should aid in identifying subunits contributing to channel function. It would be helpful to show that our method confirms previous descriptions of ciliary membrane channels. Because such reports are rare, comparison is difficult. Raychowdhury et al. Although ENaC has been found on the apical surface of cells from the inner medullary collecting duct [ 71 ], there are no reports of ENaC on the primary cilium in this portion of the nephron.

    However, the properties of TRPP2-dependent channel currents vary considerably depending on the cellular system and physiological solutions chosen [ 13 , 16 , 17 , 18 , 29 , 30 , 31 , 32 , 33 , 34 , 57 , 58 ]. Ultimately knockdown studies will be required to learn the molecular composition of the large-conductance channel in native mIMCD-3 cilia.

    Our initial studies Figure 3 indicate that the ability to record from single primary cilia may allow similar advances. In cases where primary cilia underlie the transduction of a stimulus into an electrical signal, the method reported here should allow a direct means of investigation. We describe a novel method that allows for the first time electrical recording from a freshly isolated primary cilium with minimal contributions from other cellular compartments.

    Many attempts yielded a seal with high input resistance and stability. Recording of ciliary electrical events was possible while attached to the cell and following excision of the cilium.

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    It will enable resolution of the ambiguities that inevitably result from studies in non-native membranes. Ultimately, the culturing of ciliated cells on beads may also provide a means of gathering cilia for proteomics.