Evolution: The ancient history of cilia assembly regulation.

A new study identifies a conserved regulatory mechanism for cilia assembly in the closest unicellular relatives of animals, suggesting that this mechanism was already present in a common unicellular ancestor and was repurposed during the transition to multicellularity.

Human SFI1 and Centrin form a complex critical for centriole architecture and ciliogenesis.

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.

Evolutionary conservation of centriole rotational asymmetry in the human centrosome.

Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the 'acorn.' Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly, and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.

DISCO is key to successful centriole maturation.

Centriole maturation is essential for ciliogenesis, but which proteins and how they regulate ciliary assembly is unclear. In this issue, Kumar et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202011133) shed light on this process by identifying a ciliopathy complex at the distal mother centriole that restrains centriole length and supports the formation of distal appendages.

Evolution of the centrosome, from the periphery to the center.

Centrosomes are central organelles that organize microtubules (MTs) in animals, fungi and several other eukaryotic lineages. Despite an important diversity of structure, the centrosomes of different lineages share the same functions and part of their molecular components. To uncover how divergent centrosomes are related to each other, we need to trace the evolutionary history of MT organization. Careful assessment of cytoskeletal architecture in extant eukaryotic species can help us infer the ancestral state and identify the subsequent changes that took place during evolution. This led to the finding that the last common ancestor of all eukaryotes was very likely a biflagellate cell with a surprisingly complex cytoskeletal organization. Centrosomes are likely derived from the basal bodies of such flagellate, but when and how many times this happened remains unclear. Here, we discuss different hypotheses for how centrosomes evolved in a eukaryotic lineage called Amorphea, to which animals, fungi and amoebozoans belong.

A helical inner scaffold provides a structural basis for centriole cohesion.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.

BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes. RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein. CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.

Emergence of a Bilaterally Symmetric Pattern from Chiral Components in the Planarian Epidermis.

Most animals exhibit mirror-symmetric body plans, yet the molecular constituents from which they are formed are often chiral. In planarian flatworms, centrioles are arranged in a bilaterally symmetric pattern across the ventral epidermis. Here, we found that this pattern is generated by a network of centrioles with prominent chiral asymmetric properties. We identify centriole components required for establishing asymmetric connections between centrioles and balancing their effects to align centrioles along polarity fields. SMED-ODF2, SMED-VFL1, and SMED-VFL3 affect the assembly of centriole appendages that tether cytoskeletal connectors to position the centrioles. We further show that the medio-lateral polarization of centrioles relies on mechanisms that are partly distinct on the left and right sides of the planarian body. Our findings shed light on how bilaterally symmetrical patterns can emerge from chiral cellular organizations.

Dynamic Polarization of the Multiciliated Planarian Epidermis between Body Plan Landmarks.

Polarity is a universal design principle of biological systems that manifests at all organizational scales, yet its coordination across scales remains poorly understood. Here, we make use of the extreme anatomical plasticity of planarian flatworms to probe the interplay between global body plan polarity and local cell polarity. Our quantitative analysis of ciliary rootlet orientation in the epidermis reveals a dynamic polarity field with head and tail as independent determinants of anteroposterior (A/P) polarization and the body margin as determinant of mediolateral (M/L) polarization. Mathematical modeling rationalizes the global polarity field and its response to experimental manipulations as superposition of separate A/P and M/L fields, and we identify the core PCP and Ft/Ds pathways as their molecular mediators. Overall, our study establishes a framework for the alignment of cellular polarity vectors relative to planarian body plan landmarks and establishes the core PCP and Ft/Ds pathways as evolutionarily conserved 2D-polarization module.

Processing Schmidtea mediterranea for Transmission Electron Microscopy: Classical and Microwave Techniques.

The flatworm Schmidtea mediterranea and related species are emerging model systems in such fields as stem-cell biology, regeneration, and evolutionary biology. Excellent molecular tools have been developed for S. mediterranea, but ultrastructural techniques have received less attention, which is unfortunate, as these methods are necessary to better understand the actual histological, cellular and subcellular features of regeneration and development. Tissue-processing regimens can be quite idiosyncratic for particular species or specimen types-what works for mammalian tissues or cell cultures will not necessarily give good results with freshwater planarians. Here we present detailed, optimized protocols for preparation of S. mediterranea by "standard" chemical fixation, and by a rapid microwave-based technique.

Multiciliated Cells in Animals.

Many animal cells assemble single cilia involved in motile and/or sensory functions. In contrast, multiciliated cells (MCCs) assemble up to 300 motile cilia that beat in a coordinate fashion to generate a directional fluid flow. In the human airways, the brain, and the oviduct, MCCs allow mucus clearance, cerebrospinal fluid circulation, and egg transportation, respectively. Impairment of MCC function leads to chronic respiratory infections and increased risks of hydrocephalus and female infertility. MCC differentiation during development or repair involves the activation of a regulatory cascade triggered by the inhibition of Notch activity in MCC progenitors. The downstream events include the simultaneous assembly of a large number of basal bodies (BBs)-from which cilia are nucleated-in the cytoplasm of the differentiating MCCs, their migration and docking at the plasma membrane associated to an important remodeling of the actin cytoskeleton, and the assembly and polarization of motile cilia. The direction of ciliary beating is coordinated both within cells and at the tissue level by a combination of planar polarity cues affecting BB position and hydrodynamic forces that are both generated and sensed by the cilia. Herein, we review the mechanisms controlling the specification and differentiation of MCCs and BB assembly and organization at the apical surface, as well as ciliary assembly and coordination in MCCs.

Basal bodies across eukaryotes series: basal bodies in the freshwater planarian Schmidtea mediterranea.

The freshwater planarian Schmidtea mediterranea has recently emerged as a valuable model system to study basal bodies (BBs) and cilia. Planarians are free-living flatworms that use cilia beating at the surface of their ventral epidermis for gliding along substrates. The ventral epidermis is composed of multiciliated cells (MCCs) that are similar to the MCCs in the respiratory airways, the brain ventricles, and the oviducts in vertebrates. In the planarian epidermis, each cell assembles approximately eighty cilia that beat in a coordinate fashion across the tissue. The BBs that nucleate these cilia all assemble de novo during terminal differentiation of MCCs. The genome of the planarian S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference are available. Defects induced by perturbing the expression of BB proteins can be detected simply by analyzing the locomotion of planarians. BBs are present in large numbers and in predictable orientation, which greatly facilitates analyses by immunofluorescence and electron microscopy. The great ease in targeting gene expression and analyzing associated defects allowed to identify a set of proteins required for BB assembly and function in planarian MCCs. Future technological developments, including methods for transgenic expression in planarians and in related species, will achieve turning free-living flatworms into powerful model systems to study MCCs and the associated human pathologies.

The planarian Schmidtea mediterranea as a model for studying motile cilia and multiciliated cells.

In the past few years, the freshwater planarian Schmidtea mediterranea has emerged as a powerful model system to study the assembly and function of cilia. S. mediterranea is a free-living flatworm that uses the beating of cilia on its ventral epidermis for locomotion. The ventral epidermis is composed of a single layer of multiciliated cells highly similar to the multiciliated cells that line the airway, the brain ventricles, and the oviducts in humans. The genome of S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference (RNAi) are available. Locomotion defects induced by perturbing the expression of ciliary genes can be often detected by simple visual screening, and more subtle defects can be detected by measuring locomotion speed. Cilia are present in large numbers and are directly accessible, which facilitates analyses by immunofluorescence and electron microscopy. Here we describe a set of methods for maintaining planarians in the lab. These include gene knockout by RNAi, cilia visualization by immunofluorescence, transmission electron microscopy, and live imaging.

Exploring the evolutionary history of centrosomes.

The centrosome is the main organizer of the microtubule cytoskeleton in animals, higher fungi and several other eukaryotic lineages. Centrosomes are usually located at the centre of cell in tight association with the nuclear envelope and duplicate at each cell cycle. Despite a great structural diversity between the different types of centrosomes, they are functionally equivalent and share at least some of their molecular components. In this paper, we explore the evolutionary origin of the different centrosomes, in an attempt to understand whether they are derived from an ancestral centrosome or evolved independently from the motile apparatus of distinct flagellated ancestors. We then discuss the evolution of centrosome structure and function within the animal lineage.

Analysis of ciliary assembly and function in planaria.

Planarians are free-living invertebrates that employ motile cilia for locomotion. Specifically, cilia that populate the ventral epithelium of the planarian body are highly conserved, with a 9+2 axoneme and a full complement of inner and outer arm dynein motors. The abundance of cilia on the planarian body, their unique accessibility, and high degree of conservation make this organism an attractive experimental model system for cilia biology. Moreover, planarians are genetically amenable and defects that compromise the function and structure of the cilia are not detrimental for their overall health, making them an ideal system for cilia gene loss-of-function studies. In this chapter, we provide information for introducing and maintaining planarians for experimental purposes in the laboratory and describe protocols for RNAi-induced gene knockdown studies. Furthermore, we elaborate on different imaging techniques used to analyze cilia physiology and structure, including live video microscopy, immunofluorescence analysis, and electron microscopy. Last, we provide assays for evaluating physical parameters of ciliary motility, including quantification of planarian gliding locomotion and measurement of ciliary beat frequency.

Centrosome loss in the evolution of planarians.

The centrosome, a cytoplasmic organelle formed by cylinder-shaped centrioles surrounded by a microtubule-organizing matrix, is a hallmark of animal cells. The centrosome is conserved and essential for the development of all animal species described so far. Here, we show that planarians, and possibly other flatworms, lack centrosomes. In planarians, centrioles are only assembled in terminally differentiating ciliated cells through the acentriolar pathway to trigger the assembly of cilia. We identified a large set of conserved proteins required for centriole assembly in animals and note centrosome protein families that are missing from the planarian genome. Our study uncovers the molecular architecture and evolution of the animal centrosome and emphasizes the plasticity of animal cell biology and development.

Evolution: Tracing the origins of centrioles, cilia, and flagella.

Centrioles/basal bodies (CBBs) are microtubule-based cylindrical organelles that nucleate the formation of centrosomes, cilia, and flagella. CBBs, cilia, and flagella are ancestral structures; they are present in all major eukaryotic groups. Despite the conservation of their core structure, there is variability in their architecture, function, and biogenesis. Recent genomic and functional studies have provided insight into the evolution of the structure and function of these organelles.

Building the centriole.

Centrioles are conserved microtubule-based organelles that lie at the core of the animal centrosome and play a crucial role in nucleating the formation of cilia and flagella in most eukaryotes. Centrioles have a complex ultrastructure with ninefold symmetry and a well-defined length. This structure is assembled from a host of proteins, including a variety of disease gene products. Over a century after the discovery of centrioles, the mechanisms underlying the assembly of these fascinating organelles, in particular the establishment of ninefold symmetry and the control of centriole length, are now starting to be uncovered.