The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.

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Sears PR, Yin W, Ostrowski LE. Continuous mucociliary transport by primary human airway epithelial cells in vitro.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs*13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_*343þ1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient ¼ 19.8 Hz versus control ¼ 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2 À/À mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

Airway cilia depend on precise changes in shape to transport the mucus gel overlying mucosal surfaces. The ciliary motion can be recorded in several planes using video microscopy. However, cilia are densely packed, and automated computerized systems are not available to convert these ciliary shape changes into forms that are useful for testing theoretical models of ciliary function. We developed a system for converting planar ciliary motions recorded by video microscopy into an empirical quantitative model, which is easy to use in validating mathematical models, or in examining ciliary function, e.g., in primary ciliary dyskinesia (PCD). The system we developed allows the manipulation of a model cilium superimposed over a video of beating cilia. Data were analyzed to determine shear angles and velocity vectors of points along the cilium. Extracted waveforms were used to construct a composite waveform, which could be used as a standard. Variability was measured as the mean difference in position of points on individual waveforms and the standard. The shapes analyzed were the end-recovery, end-effective, and fastest moving effective and recovery with mean (Ϯ SE) differences of 0.31(0.04), 0.25(0.06), 0.50(0.12), 0.50(0.10), m, respectively. In contrast, the same measures for three different PCD waveforms had values far outside this range. model; empirical; video; ciliated epithelia; primary ciliary dyskinesia CILIA AND FLAGELLA ARE CELLULAR organelles, which undergo dynamic cyclic shape changes that propel fluid. Most epithelial cells of the conducting airways of the lung are ciliated, and it is the function of these cells to propel mucus so that the airway is cleared of inhaled microbes and particulates. Impaired ciliary or flagellar motion is a characteristic of a number of human diseases (18,22); primary ciliary dyskinesia (PCD) and cystic fibrosis typify failures of ciliary function as a result of defects intrinsic to cilia and the result of the ciliary environment, respectively.Because the geometry of dynein-tubulin interactions and the periodic nature of dynein activation are lost when the axoneme is dismantled, it is not possible to elucidate the mechanism generating the dynamics from experiments with isolated dynein motors processing on microtubules. It is necessary to study the dynamics directly. For flagella, clear video images allow the accurate recording organelle shapes by hand and the derivation of parameters such as curvature (17,23). Automated video analysis that can be used to digitize flagellar shapes and extract dynamics has been demonstrated by Baba and Mogami (1). Given the symmetry of the flagellar beat, Eshel and Brokaw (9) showed that recording shear angle from video frames enabled the fitting of the dynamics over many cycles to a trigonometric function, thus allowing the dynamics to be described with a smooth parameter set. Some theoretical dynamics have been coupled to the experimentally acquired dynamics. For example, the shear angle of the flagellar dynamics has been used to analyze dyn...

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.

Author contributions R.R.C. initiated the project, phenotyped the index proband, designed and performed all experiments except as detailed herewith, analyzed data, prepared figures, and wrote the manuscript. D.T.M assisted with designing and performing cell culture, IF, and FACS experiments, analyzed data, and edited the manuscript. H.M.L. performed μOCT experiments, analyzed data, and prepared figures. J.Y. assisted with molecular cloning, site-directed mutagenesis, and cell culture and edited the manuscript. H.E.S. performed whole exome sequencing and linkage analysis on kindreds 1-3. M.S.T. acquired clinical histopathology images and prepared figures. G.W.D. performed sequencing, molecular biology, and HSVM analysis on kindred 4. M.A.Z. led molecular analysis of kindred 5. J.C. performed and interpreted kindred 5 ciliary EM and HSVM. L.A.D. identified kindred 5 patients and provided clinical data. P.S. performed HSVM. K.E.B. and L.P.H. assisted with obtaining proband 1 clinical samples. I.A. identified bronchiectasis kindreds 2 and 3. E.M.F. assisted with analysis of phosphoproteomics data. V.V. assisted with IF experiments, analyzed data, and edited the manuscript. H.O. supervised and led kindred 4 analyses. M.R.K. supervised and led kindred 5 molecular analysis and clinical phenotyping. G.J.T. supervised μOCT experiments and analyzed data. F.S.A. supervised whole exome sequencing and linkage analysis of kindreds 1-3, analyzed genetics data, and edited the manuscript. D.M.S. supervised the project, designed experiments, and edited the manuscript.

Prior papers have introduced steerable needles composed of precurved concentric tubes. The curvature and extent of these needles can be controlled by the relative rotation and translation of the individual tubes. Under certain assumptions on the geometry and design of these needles, the forward kinematics problem can be solved in closed form by means of algebraic equations. The inverse kinematics problem, however, is not as straightforward owing to the nonlinear map between relative tube displacements and needle tip configuration as well as to the multiplicity of solutions as the number of tubes increases. This paper presents a general approach to solving the inverse kinematics problem using a pseudoinverse solution together with gradients of nullspace potential functions to enforce geometric and mechanical constraints.