Basal body organization and cell geometry during the cell cycle in Tetrahymena thermophila.

Tetrahymena thermophila possesses arrays of motile cilia that promote fluid flow for cell motility. These consist of intricately organized basal bodies (BBs) that nucleate and position cilia at the cell cortex. Tetrahymena cell geometry and spatial organization of BBs play important roles in cell size, swimming, feeding, and division. How cell geometry and BB organization are established and maintained remains poorly understood, and prior studies have been limited due to difficulties in accurate BB identification and small sample size. We therefore developed an automated image processing pipeline that segments single cells, distinguishes unique BB populations, assigns BBs into distinct ciliary rows, and distinguishes new from mature BBs. We identified unique features to describe the variation of cell shape and BB spatial organization in unsynchronized single-cell images. The results reveal asymmetries in BB distribution and ingression of the cytokinetic furrow within the cell. Moreover, we establish novel spatial and temporal waves in new BB assembly through the cell cycle. Finally, we used measurements from single cells across the cell cycle to construct a generative model that allows synthesis of movies depicting single cells progressing through the cell cycle. Our approach is expected to be of particular value for characterizing Tetrahymena mutants.

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.

Ciliate cortical organization and dynamics for cell motility: Comparing ciliates and vertebrates.

The generation of efficient fluid flow is crucial for organismal development and homeostasis, sexual reproduction, and motility. Multi-ciliated cells possess fields of motile cilia that beat in synchrony to propel fluid. Ciliary arrays are remarkably conserved in their organization and function. Ciliates have polarized multi-ciliary arrays (MCAs) to promote fluid flow for cell motility. The ciliate cortex is decorated with hundreds of basal bodies (BB) forming linear rows along the cell's anterior-posterior axis. BBs scaffold and position cilia to form the organized ciliary array. Nascent BBs assemble at the base of BBs. As nascent BBs mature, they integrate into the cortical BB and cytoskeletal network and nucleate their own cilium. The organization of MCAs is balanced between cortical stability and cortical dynamism. The cortical cytoskeletal network both establishes and maintains a stable organization of the MCA in the face of mechanical forces exerted by ciliary beating. At the same time, MCA organization is plastic, such that it remodels for optimal ciliary mobility during development and in response to environmental conditions. Such plasticity promotes effective feeding and ecological behavior required for these organisms. Together, these properties allow an organism to effectively sense, adapt to, and move through its environment.

RAB19 Directs Cortical Remodeling and Membrane Growth for Primary Ciliogenesis.

Primary cilia are sensory organelles that utilize the compartmentalization of membrane and cytoplasm to communicate signaling events, and yet, how the formation of a cilium is coordinated with reorganization of the cortical membrane and cytoskeleton is unclear. Using polarized epithelia, we find that cortical actin clearing and apical membrane partitioning occur where the centrosome resides at the cell surface prior to ciliation. RAB19, a previously uncharacterized RAB, associates with the RAB-GAP TBC1D4 and the HOPS-tethering complex to coordinate cortical clearing and ciliary membrane growth, which is essential for ciliogenesis. This RAB19-directed pathway is not exclusive to polarized epithelia, as RAB19 loss in nonpolarized cell types blocks ciliogenesis with a docked ciliary vesicle. Remarkably, inhibiting actomyosin contractility can substitute for the function of the RAB19 complex and restore ciliogenesis in knockout cells. Together, this work provides a mechanistic understanding behind a cytoskeletal clearing and membrane partitioning step required for ciliogenesis.

Ciliary force-responsive striated fibers promote basal body connections and cortical interactions.

Multi-ciliary arrays promote fluid flow and cellular motility using the polarized and coordinated beating of hundreds of motile cilia. Tetrahymena basal bodies (BBs) nucleate and position cilia, whereby BB-associated striated fibers (SFs) promote BB anchorage and orientation into ciliary rows. Mutants that shorten SFs cause disoriented BBs. In contrast to the cytotaxis model, we show that disoriented BBs with short SFs can regain normal orientation if SF length is restored. In addition, SFs adopt unique lengths by their shrinkage and growth to establish and maintain BB connections and cortical interactions in a ciliary force-dependent mechanism. Tetrahymena SFs comprise at least eight uniquely localizing proteins belonging to the SF-assemblin family. Loss of different proteins that localize to the SF base disrupts either SF steady-state length or ciliary force-induced SF elongation. Thus, the dynamic regulation of SFs promotes BB connections and cortical interactions to organize ciliary arrays.

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.