Embryonic Node Research Articles

Advances in biomechanical studies of nodal ciliary movement

In recent years, cilia have become a research hotspot in the biomedicine field. As the important functional organelles of cells, cilia can not only perceive extracellular mechanical and chemical signals, but also transduce signals to initiate cellular responses. Based on ultrastructural and functional differences, cilia can be classified as immotile cilia or motile cilia. The immotile cilia are also called primary cilia, and its principal functions are sensing cellular antennae and transducing various morphogenetic and sensory signals. By contrast, motile cilia are usually present on a cell’s surface, and the presence of dynein arms confers them with the ability to beat. Even though exteriorly the vertebrate body is bilaterally symmetric, the internal organs and associated vasculature show a clear left-right (L-R) asymmetry in the placement and patterning. L-R asymmetries in embryos occur earlier than visceral organs. Thus, for bilaterally symmetric embryo, it is a challenging problem to distinguish left from right. In the early stage of vertebrate embryonic development, there are several rotating monocilia in the embryonic node, with a 9+0 ultrastructure. Namely, the main composition of the axoneme is represented by nine surrounding doublet microtubules. The dynein in the axoneme forms bridges among neighbouring microtubule doublets, and it provides the power and control for cilia movement through ATP hydrolysis. Then, the nodal cilia rotate to drive the surrounding fluid to flow leftward, which causes the embryo to sense the left and right axis directions. Beyond that, abnormalities in the structure and function of nodal cilia can lead to serious developmental disorders. Thus, the study of nodal cilia contributes to our understanding in the developmental processes of vertebrates. The connection between cilia and L–R asymmetry originated in the mid-1970s. Through the studies of Kartagener syndrome, Afzelius and Pedersen found that the Autosomal recessive genetic disorder was caused by immotile cilia. From then on, more and more researchers have studied cilia in many aspects. Although the understanding of the structure and function of nodal cilia continues to deepen, the internal mechanism of cilia movement and the mechanism of nodal flow propagation remain questionable. This paper will summarize the progress of these studies from the molecular, cellular, and extracellular fluid levels. Above all, the related researches on cilia structure, dynein structure, and numerical simulation of dynein activity model are introduced. Furthermore, based on morphogen hypothesis, nodal vesicular parcel (NVP) hypothesis and two-cilia hypothesis, the role of nodal cilia in embryonic development and the effect of cilia position and state on pericapillary fluids are described. After that, the necessity of unidirectional nodal flow for embryo development is introduced from the aspects of transmitting small molecule signals and generating the mechanical force. At the same time, several methods for measuring flow velocity are introduced. Finally, the problems to be solved in the biomechanical research of ciliary movement are summarized and prospected. Cross-scale simulation analysis can break the inherent limitations of single-scale magnitude. In the future, research work can be carried out in the following aspects: improving experimental observation techniques, measuring the precise properties of the nodal cilia, and optimizing simulation models.

Knockdown of Mns1 Increases Susceptibility to Craniofacial Defects Following Gastrulation-Stage Alcohol Exposure in Mice.

MNS1 (meiosis-specific nuclear structural protein 1) is necessary for motile cilia function, such as sperm flagella or those found in the embryonic primitive node. While little is known regarding the function or expression pattern of MNS1 in the embryo, co-immunoprecipitation experiments in sperm have determined that MNS1 interacts with ciliary proteins, which are also important during development. Establishment of morphogenic gradients is dependent on normal ciliary motion in the primitive node beginning during gastrulation (gestational day [GD] 7 in the mouse, second-third week of pregnancy in humans), a critical window for face, eye, and brain development and particularly susceptible to perturbations of developmental signals. The current study investigates the role of Mns1 in craniofacial defects associated with gastrulation-stage alcohol exposure. On GD7, pregnant Mns1+/- dams were administered 2 doses of ethanol (5.8g/kg total) or vehicle 4hours apart to target gastrulation. On GD17, fetuses were examined for ocular defects by scoring each eye on a scale from 1 to 7 (1=normal, 2 to 7=defects escalating in severity). Craniofacial and brain abnormalities were also assessed. Prenatal alcohol exposure (PAE) significantly increased the rate of defects in wild-type fetuses, as PAE fetuses had an incidence rate of 41.18% compared to a 10% incidence rate in controls. Furthermore, PAE interacted with genotype to significantly increase the defect rate and severity in Mns1+/- (64.29%) and Mns1-/- mice (92.31%). PAE Mns1-/- fetuses with severe eye defects also presented with craniofacial dysmorphologies characteristic of fetal alcohol syndrome and midline tissue loss in the brain, palate, and nasal septum. These data demonstrate that a partial or complete knockdown of Mns1 interacts with PAE to increase the susceptibility to ocular defects and correlating craniofacial and brain anomalies, likely though interaction of alcohol with motile cilia function. These results further our understanding of genetic risk factors that may underlie susceptibility to teratogenic exposures.

Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.

Biological fluid mechanics under the microscope: a tribute to John Blake

John Blake (1947–2016) was a leader in fluid mechanics, his two principal areas of expertise being biological fluid mechanics on microscopic scales and bubble dynamics. He produced leading research and mentored others in both Australia, his home country, and the UK, his adopted home. This article reviews John Blake’s contributions in biological fluid mechanics, as well as gives the author’s personal viewpoint as one of the many graduate students and researchers who benefitted from his supervision, guidance and inspiration. The key topics from biological mechanics discussed are: “squirmer” models of protozoa, the method of images in Stokes flow and the “blakelet” solution, discrete cilia modelling via slender body theory, physiological flows in respiration and reproduction, blinking stokeslets in microorganism feeding, human sperm motility and embryonic nodal cilia. doi:10.1017/S1446181118000020

BIOLOGICAL FLUID MECHANICS UNDER THE MICROSCOPE: A TRIBUTE TO JOHN BLAKE

John Blake (1947–2016) was a leader in fluid mechanics, his two principal areas of expertise being biological fluid mechanics on microscopic scales and bubble dynamics. He produced leading research and mentored others in both Australia, his home country, and the UK, his adopted home. This article reviews John Blake’s contributions in biological fluid mechanics, as well as gives the author’s personal viewpoint as one of the many graduate students and researchers who benefitted from his supervision, guidance and inspiration. The key topics from biological mechanics discussed are: “squirmer” models of protozoa, the method of images in Stokes flow and the “blakelet” solution, discrete cilia modelling via slender body theory, physiological flows in respiration and reproduction, blinking stokeslets in microorganism feeding, human sperm motility and embryonic nodal cilia.

The Inv compartment of renal cilia is an intraciliary signal-activating center to phosphorylate ANKS6

Connections between cilia and renal cystic diseases are well known, yet molecular mechanisms remain undefined. Cysto-proteins localized in the Inv compartment of cilia (INV, NPHP3, NEK8, and ANKS6) constitute a distinct group. Here we created and analyzed mutant mice (G2A mice) with a defective cilia localization signal in the Nphp3 gene. Mutant NPHP3 was absent the binding capacity of UNC119, a carrier protein responsible for the delivery of myristoylated cargo to the cilium, so ciliary localization was reduced or lost in the kidney but not in the embryonic node. Mutant mice developed renal cysts but not situs abnormalities. Although ciliary localization of INV, NEK8, and ANKS6 did not change in the kidneys of Nphp3 mutant mice, ANKS6 phosphorylation was impaired. In general, ANKS6 levels decrease with age in the kidneys of wild-type mice. However, cystic kidneys in G2A and Inv mice maintained high levels of a non-phosphorylated form of ANKS6. We found INV and NPHP3 cooperate and promote ANKS6 phosphorylation by NEK8 in renal cilia. Thus, there is a novel signaling path from cilia in which ANKS6 functions as a signal mediator and link between cilia and the cytoplasm to regulate kidney morphogenesis.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer’s vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

1700012B09Rik, a FOXJ1 effector gene active in ciliated tissues of the mouse but not essential for motile ciliogenesis

In humans and mice, motile cilia occur on the surface of the embryonic ventral node, on respiratory and ependymal epithelia and in reproductive organs where they ensure normal left-right asymmetry of the organism, mucociliary clearance of airways, homeostasis of the cerebrospinal fluid and fertility. The genetic programme for the formation of motile cilia, thus critical for normal development and health, is switched on by the key transcription factor FOXJ1. In previous microarray screens for murine FOXJ1 effectors, we identified candidates for novel factors involved in motile ciliogenesis, including both genes that are well conserved throughout metazoa and beyond, like FOXJ1 itself, and genes without overt homologues outside higher vertebrates.Here we examine one of the novel murine FOXJ1 effectors, the uncharacterised 1700012B09Rik whose homologues appear to be restricted to higher vertebrates. In mouse embryos and adults, 1700012B09Rik is predominantly expressed in motile ciliated tissues in a FOXJ1-dependent manner. 1700012B09RIK protein localises to basal bodies of cilia in cultured cells. Detailed analysis of 1700012B09RiklacZ knock-out mice reveals no impaired function of motile cilia or non-motile cilia. In conclusion, this novel FOXJ1 effector is associated mainly with motile cilia but – in contrast to other known FOXJ1 targets – its putative ciliary function is not essential for development or health in the mouse, consistent with a late emergence during evolution of motile ciliogenesis.

Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans.

Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1. DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans.

Mutations in Dnaaf1 and Lrrc48 Cause Hydrocephalus, Laterality Defects, and Sinusitis in Mice.

We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48. Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.

Primary cilia are not calcium-responsive mechanosensors.

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium1. This Ca2+- Responsive MechanoSensor (CaRMS) hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers2,3. Here, we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. First, we developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator (GECI) in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca2+ influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signaling.

ZMYND10--Mutation Analysis in Slavic Patients with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare recessive disease with a prevalence of 1/10,000; its symptoms are caused by a kinetic dysfunction of motile cilia in the respiratory epithelium, flagella in spermatozoids, and primary cilia in the embryonic node. PCD is genetically heterogeneous: genotyping the already known PCD-related genes explains the genetic basis in 60–65% of the cases, depending on the population. While identification of new genes involved in PCD pathogenesis remains crucial, the search for new, population-specific mutations causative for PCD is equally important. The Slavs remain far less characterized in this respect compared to West European populations, which significantly limits diagnostic capability. The main goal of this study was to characterize the profile of causative genetic defects in one of the PCD-causing genes, ZMYND10, in the cohort of PCD patients of Slavic origin. The study was carried out using biological material from 172 unrelated PCD individuals of Polish origin, with no causative mutation found in nine major PCD genes. While none of the previously described mutations was found using the HRM-based screening, a novel frameshift mutation (c.367delC) in ZMYND10, unique for Slavic PCD population, was found in homozygous state in two unrelated PCD patients. Immunofluorescence analysis confirmed the absence of outer and inner dynein arms from the ciliary axoneme, consistent with the already published ZMYND10-mutated phenotype; cDNA analysis revealed the lack of ZMYND10 mRNA, indicating nonsense-mediated decay of the truncated transcript.

The primary cilium is a self-adaptable, integrating nexus for mechanical stimuli and cellular signaling.

ABSTRACTMechanosensation is crucial for cells to sense and respond to mechanical signals within their local environment. While adaptation allows a sensor to be conditioned by stimuli within the environment and enables its operation in a wide range of stimuli intensities, the mechanisms behind adaptation remain controversial in even the most extensively studied mechanosensor, bacterial mechanosensitive channels. Primary cilia are ubiquitous sensory organelles. They have emerged as mechanosensors across diverse tissues, including kidney, liver and the embryonic node, and deflect with mechanical stimuli. Here, we show that both mechanical and chemical stimuli can alter cilium stiffness. We found that exposure to flow stiffens the cilium, which deflects less in response to subsequent exposures to flow. We also found that through a process involving acetylation, the cell can biochemically regulate cilium stiffness. Finally, we show that this altered stiffness directly affects the responsiveness of the cell to mechanical signals. These results demonstrate a potential mechanism through which the cell can regulate its mechanosensing apparatus.

A human laterality disorder caused by a homozygous deleterious mutation in MMP21

BackgroundLaterality in the vertebrate embryo is determined by left-right asymmetric gene expression driven by the flow of extraembryonic fluid across the embryonic node. Defects in these processes cause heterotaxy, the...

A novel form of PCD that impacts nodal, but not tracheal cilia

Motile cilia, in the embryonic node, drive a leftward fluid flow (termed nodal flow) that establishes the left-right axis. We identified lrm5 in a genetic screen for mouse left-right patterning mutants - the embryos exhibited disturbed situs. Mapping and sequencing revealed a novel mutation in the axonemal dynein heavy chain locus Dnah11: loss of function gives rise to immotile cilia in mice; many human primary ciliary dyskinesia (PCD) patients with DNAH11 mutations have hyper-motile cilia. To our surprise, unlike the previously characterised Dnah11iv mutant, lrm5 tracheal ciliary beat frequency (CBF) was normal. However, the number of lrm5 homozygotes at weaning was lower than Mendelian ratios would predict. Age of death analysis identified a 50% reduction in embryos between E14.5 and E15.5, consistent with death from embryonic cardiac failure. Expression analysis of early molecular markers of left-right asymmetry revealed randomised or bilateral activation of the normally left-sided Nodal Cascade. As this suggested an early, primary patterning defect, we analysed nodal flow by particle image velocimetry (PIV); rather than the wild-type leftward flow, or the absent flow in Dnah11iv, we observed a chaotic fluid flow in lrm5 nodes. We therefore assessed nodal CBF and ciliary amplitude by DIC microscopy; lrm5 cilia beat at 1.5x normal frequency, but with an abnormal motion. In summary, lrm5 is a novel form of PCD, impacting nodal but not tracheal ciliary beating. We would predict that equivalent mutations in humans might underlie situs defects and congenital heart disease in the absence of respiratory disease.

Investigation into the Importance of genes encoding ciliary proteins in congenital heart disease using whole exome sequencing

Congenital Heart Disease (CHD) is the most common congenital defect. Many families with left-right laterality defects and complex CHD have an unknown genetic aetiology. Many ciliopathies, including Primary Ciliary Dyskinesia (PCD), are associated with intracardiac defects. The role of primary cilia in cardiac morphogenesis remains unknown, although cardiac cilia have roles that are distinct from the definition of laterality at the embryonic node. We hypothesise that defects in genes important in the assembly and function of cilia are responsible for some inherited forms of CHD. This research project aims to recruit families with a recurrence of CHD and to perform Whole Exome Sequencing (WES) to identify putative pathogenic variants and to delineate novel genetic causes of CHD. Twelve families have now been recruited and WES has been carried out in seven of these families using paired-end sequencing. Data analysis follows a standardised pipeline to call and then filter variants in order to assess their pathogenic potential. Variants are prioritized on the basis of known or suspected function of the encoded protein, and publicly-available RNA expression data. Variant filtering has allowed the identification of a limited number of candidate variants in recruited families. Of particular interest, a likely causative homozygous variant within a PCD gene has been identified in two siblings affected with heterotaxy, thus confirming a link between ciliopathies and CHD. The function and interactions of identified genes will be assessed, using cellular techniques and animal models, to provide insights into the pathogenesis of CHD.

Identification of Polycystin-2 and CFTR common targets

Autosomal-Dominant-Polycystic-Kidney-Disease type-II (ADPKD-II) is caused by mutations in the Polycystin-2 (PC2) encoding gene. The dysfunction of this Ca2+-conducting channel leads to the formation of fluid-filled renal cysts (1). Through poorly known mechanisms, cystogenesis entails an overstimulation of Cystic-Fibrosis-Transmembrane-conductance-Regulator (CFTR), a key Cl--channel in epithelia's ion/fluid transport (2). To study PC2-CFTR crosstalk we used the zebrafish embryonic node (Kupffer's Vesicle, KV) as a model system. Both proteins are required for KV proper function (3,4).

Motile and Sensory Cilia Function in Zebrafish

Advances in developmental genetics and human disease gene cloning have highlighted the essential roles played by cilia in developmental cell fate decisions, left–right asymmetry, and the pathology of human congenital disorders. Cilia-driven fluid flow in the embryonic node highlights the role of motile cilia in the generation of mechanical signals in development. Sensory cilia in photoreceptors and olfactory neurons highlight the role of cilia in phototransduction and G-protein coupled receptor signaling. Using a zebrafish knockdown screen of cilia candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility and generation of proper left-right asymmetry. c21orf59 and c15orf26 knockdown in zebrafish blocked outer dynein arm assembly, while ccdc65 knockdown altered cilia beat pattern. To study sensory cilia function we generated a new cilia assay based on calcium biosensor (GCaMP5) expression in zebrafish ciliated olfactory neurons (OSNs). Ciliated OSNs are activated by bile acid olfactory receptors on outward facing terminal sensory cilia. We found that ciliated OSNs responded with a robust fluorescence signal to specific odorants and that the cilia mutant fish oval, (ift88-/-) and ift172 knockdown larvae showed severely shortened OSN cilia and markedly reduced responses to bile acids while other odorant responses were unaffected. In summary, the zebrafish provides a robust platform for cilia gene discovery, in vivo functional assays of cilia mutant allele strength, and mechanisms of sensory cilia assembly and function. Supported by NIH grant DK053093

The Primary Cilium is a Self-Adaptable, Integrating Nexus for Mechanical Stimuli and Cell Signaling

Mechanosensation is critical for cells to maintain homeostasis and in devastating diseases, including atherosclerosis, osteoporosis and cancer. Although several cellular mechanosensing structures have been described, none have been shown to adapt to mechanical stimuli or be regulated in non-excitable cells. Primary cilia are ubiquitous chemo-mechanical sensors and function as mechanosensors in several tissues, including kidney, liver, cartilage, bone, and the embryonic node, deflecting in response to mechanical stimuli. Several groups have shown cilia length adapts in response to mechanical stimuli while others have shown relatively small changes in length can affect cilia deflection and downstream load-induced changes in gene and protein expression. Collectively, this suggests cilia mechanosensitivity may be modulated. Here, we show that both mechanical and chemical mechanisms can alter ciliary rigidity. We exposed mouse inner medullary collecting duct cells transfected with a live-cell marker for primary cilia to flow. Cilium bending behavior was captured with high-speed confocal microscopy and modeled as a beam anchored by a torsional spring. We found exposure to flow stiffened the cilium up to 4-fold (n=12), deflecting less in response to subsequent exposures to flow. We hypothesized that modifying tubulin may mimic this stiffening. Post-translational modifications of tubulin, such as acetylation, have been shown to stiffen microtubules. Interestingly, acetylation can increase with mechanical stimuli. Using a potent pharmaceutical agent and a siRNA knockdown to alter acetylation, we showed that through acetylation the cell can biochemically regulate ciliary stiffness up to 4-fold (n=5/group). We further showed that this altered stiffness directly affects the sensitivity of the cell to mechanical signals, resulting in a 2-fold change in gene expression (n=5/group). We demonstrated, for the first time, a potential mechanism through which the cell can regulate its mechanosensing apparatus.

Microscale imaging of cilia-driven fluid flow.

Cilia-driven fluid flow is important for multiple processes in the body, including respiratory mucus clearance, gamete transport in the oviduct, right-left patterning in the embryonic node, and cerebrospinal fluid circulation. Multiple imaging techniques have been applied toward quantifying ciliary flow. Here, we review common velocimetry methods of quantifying fluid flow. We then discuss four important optical modalities, including light microscopy, epifluorescence, confocal microscopy, and optical coherence tomography, that have been used to investigate cilia-driven flow.