Basal bodies bend in response to ciliary forces.

Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia.

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell's cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.

Plastic cell morphology changes during dispersal.

Dispersal is the movement of organisms from one habitat to another that potentially results in gene flow. It is often plastic, allowing organisms to adjust dispersal movements depending on environmental conditions. A fundamental aim in ecology is to understand the determinants underlying dispersal and its plasticity. We utilized 22 strains of the ciliate Tetrahymena thermophila to determine if different phenotypic dispersal strategies co-exist within a species and which mechanisms underlie this variability. We quantified the cell morphologies impacting cell motility and dispersal. Distinct differences in innate cellular morphology and dispersal rates were detected, but no universally utilized combinations of morphological parameters correlate with dispersal. Rather, multiple distinct and plastic morphological changes impact cilia-dependent motility during dispersal, especially in proficient dispersing strains facing challenging environmental conditions. Combining ecology and cell biology experiments, we show that dispersal can be promoted through plastic motility-associated changes to cell morphology and motile cilia.

Microtubule glycylation promotes attachment of basal bodies to the cell cortex.

Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.

Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces.

Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms.

Human amnion mesenchymal cells are pro-inflammatory when activated by the Toll-like receptor 2/6 ligand, macrophage-activating lipoprotein-2.

INTRODUCTION: Infection accounts for over 40% of preterm premature rupture of the fetal membranes (PPROM), a major cause of preterm birth. Toll-like receptors (TLR) play key roles in pathogen surveillance but their expression and function in amnion mesenchymal cells (AMC) is unclear. The aims of this study were to determine the expression of all TLR isoforms and the effect of macrophage-activating lipoprotein-2 (MALP-2), derived from a common pathogen involved in PPROM, on human AMC. METHODS: AMC were isolated from normal, term amnion from repeat cesarean section. Semi-quantitative RT-PCR, immunocytochemistry, immunohistochemistry and western blotting were used to detect TLR isoform expression. Immunocytochemistry of NF-κB p65, pro-inflammatory cytokine secretion (ELISA), MTT assay, LDH assay, immunoblotting of cytosolic cytochrome c and cleaved caspase-3, and expression of 84 microRNAs by Qiagen miRNA PCR array were used to determine the functional effect of MALP-2 on AMC. RESULTS: TLR1-10 was detected in AMC, and protein expression of TLR2, 4, and 6 were confirmed. MALP-2 induced nuclear translocation of p65, reaching significance after 45 min (ANOVA, P < 0.05). MALP-2 did not cause apoptosis but did lead to significant secretion of IL-4, IL-6, and IL-8 (P < 0.05, 0.01, 0.001, respectively) and significant changes in miRNA-320a and miRNA-18a (P < 0.05). DISCUSSION: These results suggest that AMC elicit a pro-inflammatory response following stimulation with the known TLR2/6 ligand MALP-2. This data supports the idea that AMC express the innate immune system receptors that could help with immune surveillance during infection and contribute to inflammatory responses that lead to PPROM.