Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Comparative Super-Resolution Mapping of Basal Feet Reveals a Modular but Distinct Architecture in Primary and Motile Cilia.

In situ molecular architecture analysis of organelles and protein assemblies is essential to understanding the role of individual components and their cellular function, and to engineering new molecular functionalities. Through a super-resolution-driven approach, here we characterize the organization of the ciliary basal foot, an appendage of basal bodies whose main role is to provide a point of anchoring to the microtubule cytoskeleton. Quantitative image analysis shows that the basal foot is organized into three main regions linked by elongated coiled-coil proteins, revealing a conserved modular architecture in primary and motile cilia, but showing distinct features reflecting its specialized functions. Using domain-specific BioID proximity labeling and super-resolution imaging, we identify CEP112 as a basal foot protein and other candidate components of this assembly, aiding future investigations on the role of basal foot across different cilia systems.

A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies.

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients' cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

Author Correction: A novel atypical sperm centriole is functional during human fertilization.

In the original version of this Article, the affiliation details for Jadranka Loncarek and Vito Mennella were incorrectly given as 'Cell Biology Program, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, ON, M5G 1X8, Canada' and 'Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, 1050 Boyles Street, Frederick, MD, 21702, USA', respectively. This has now been corrected in both the PDF and HTML versions of the Article.

A novel atypical sperm centriole is functional during human fertilization.

The inheritance of the centrosome during human fertilization remains mysterious. Here we show that the sperm centrosome contains, in addition to the known typical barrel-shaped centriole (the proximal centriole, PC), a surrounding matrix (pericentriolar material, PCM), and an atypical centriole (distal centriole, DC) composed of splayed microtubules surrounding previously undescribed rods of centriole luminal proteins. The sperm centrosome is remodeled by both reduction and enrichment of specific proteins and the formation of these rods during spermatogenesis. In vivo and in vitro investigations show that the flagellum-attached, atypical DC is capable of recruiting PCM, forming a daughter centriole, and localizing to the spindle pole during mitosis. Altogether, we show that the DC is compositionally and structurally remodeled into an atypical centriole, which functions as the zygote's second centriole. These findings now provide novel avenues for diagnostics and therapeutic strategies for male infertility, and insights into early embryo developmental defects.