Hereditary Mucin Deficiency Caused by Biallelic Loss of Function of MUC5B.

Rationale: Mucin homeostasis is fundamental to airway health. Upregulation of airway mucus glycoprotein MUC5B is observed in diverse common lung diseases and represents a potential therapeutic target. In mice, Muc5b is required for mucociliary clearance and for controlling inflammation after microbial exposure. The consequences of its loss in humans are unclear. Objectives: The goal of this study was to identify and characterize a family with congenital absence of MUC5B protein. Methods: We performed whole-genome sequencing in an adult proband with unexplained bronchiectasis, impaired pulmonary function, and repeated Staphylococcus aureus infection. Deep phenotyping over a 12-year period included assessments of pulmonary radioaerosol mucociliary clearance. Genotyping with reverse phenotyping was organized for eight family members. Extensive experiments, including immunofluorescence staining and mass spectrometry for mucins, were performed across accessible sample types. Measurements and Main Results: The proband, and her symptomatic sibling who also had extensive sinus disease with nasal polyps, were homozygous for a novel splicing variant in the MUC5B gene (NM_002458.2: c.1938 + 1G>A). MUC5B was absent from saliva, sputum, and nasal samples. Mucociliary clearance was impaired in the proband, and large numbers of apoptotic macrophages were present in sputum. Three siblings heterozygous for the familial MUC5B variant were asymptomatic but had a shared pattern of mild lung function impairments. Conclusions: Congenital absence of MUC5B defines a new category of genetic respiratory disease. The human phenotype is highly concordant with that of the Muc5b-/- murine model. Further study of individuals with decreased MUC5B production could provide unique mechanistic insights into airway mucus biology.

Patterning of Structurally Anisotropic Composite Hydrogel Sheets.

Compositional and structural patterns play a crucial role in the function of many biological tissues. In the present work, for nanofibrillar hydrogels formed by chemically cross-linked cellulose nanocrystals (CNC) and gelatin, we report a microextrusion-based 3D printing method to generate structurally anisotropic hydrogel sheets with CNCs aligned in the direction of extrusion. We prepared hydrogels with a uniform composition, as well as hydrogels with two different types of compositional gradients. In the first type of gradient hydrogel, the composition of the sheet varied parallel to the direction of CNC alignment. In the second hydrogel type, the composition of the sheet changed orthogonally to the direction of CNC alignment. The hydrogels exhibited gradients in structure, mechanical properties, and permeability, all governed by the compositional patterns, as well as cytocompatibility. These hydrogels have promising applications for both fundamental research and for tissue engineering and regenerative medicine.

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.

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.