Journal of cell science最新文献

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogenous disorder resulting from dysfunctional motile cilia that is characterized by chronic, progressive lung disease with currently no corrective therapies available. Here, we test the efficacy of selective organ targeting lipid nanoparticles (SORT-LNPs) that were optimized for potency and delivery to respiratory cells containing an mRNA encoding an axonemal protein to rescue ciliary activity in a murine culture model of PCD. Utilizing murine nasopharyngeal epithelial cell (mNPEC) cultures isolated from a conditional Dnai1 knockout mouse model of the known human PCD-associated gene DNAI1 homolog, we tested if SORT-LNPs containing an optimized Dnai1 mRNA could rescue ciliary activity. Treatment of differentiating and well-differentiated Dnai1 knockout mNPECs with SORT-LNP-Dnai1 mRNA led to a dose-dependent increase in levels of DNAI1 protein and incorporation into ciliary axonemes, resulting in rescued ciliary activity with normal ciliary beat frequency that persisted for over 3 weeks. These data support further clinical development of an mRNA-based therapeutic with LNP-mediated delivery as a treatment for individuals with PCD with disease-causing DNAI1 mutations.

The microtubule motor dynein-2 is responsible for retrograde intraflagellar transport (IFT), a process crucial for cilia assembly and cilium-dependent signaling. Mutations in genes encoding dynein-2 subunits interfere with ciliogenesis and are among the most frequent causes of skeletal ciliopathies. Despite its importance, little is known regarding dynein-2 assembly and regulation. Here, we identify the molecular HSP90 chaperone as an essential regulator of dynein-2 complex stability and function. Pharmacological inhibition of HSP90 causes a severe decrease in the levels of dynein-2 subunits, without detectable alterations in cytoplasmic dynein-1 and the anterograde IFT kinesin-2 motor KIF3A. Consistent with disrupted dynein-2 function, HSP90 inhibition progressively disrupts retrograde IFT and severely impairs ciliogenesis. We demonstrate that HSP90 associates with the dynein-2 complex, promoting its assembly and stabilization. These results establish dynein-2 as an HSP90 client and provide important mechanistic insights into the regulation of dynein-2 assembly.

Primary cilia are microtubule-based sensory organelles that have been conserved throughout eukaryotic evolution. As discussed in this Review, a cilium is an elongated and highly specialized structure, and, together with its ability to selectively traffic and concentrate proteins, lipids and second messengers, it creates a signaling environment distinct from the cell body. Ciliary signaling pathways adopt a bow-tie network architecture, in which diverse inputs converge on shared effectors and second messengers before diverging to multiple outputs. Unlike other cellular bow-tie systems, cells exploit ciliary geometry, compartmentalization and infrastructure to enhance sensitivity at multiple scales, from individual molecular reactions to entire signaling pathways. In cilia, integration of the bow-tie network architecture with their specialized structure and unique environment confers robustness and evolvability, which enables cilia to acquire diverse signaling roles. However, this versatility comes with vulnerability - rare mutations that disrupt the features most essential for cilia robustness cause multisystem ciliopathies.

Motile cilia are microtubule-based organelles that generate fluid flow through coordinated beating, a process powered by axonemal dynein motors. Dyneins are pre-assembled in the cytoplasm by a suite of proteins called dynein axonemal assembly factors (DNAAFs). Genetic variants affecting either the motors or the assembly factors cause motile ciliopathy. In recent years, DNAAFs have been found to function in conjunction with heat-shock protein (HSP) chaperone systems and organize with dynein subunits within cytoplasmic foci known as 'dynein axonemal particles' (DynAPs). In this Perspective, we provide our view on the assembly and potential function of DynAPs, as well as their place within the broader context of motile ciliated cells.

Motile cilia are highly specialized organelles that generate rhythmic beating to drive fluid flow and cell movement. This activity depends on the unique molecular machinery of the axoneme, which is composed of hundreds of proteins that operate in a highly coordinated manner. Recent advances in cryo-electron microscopy have uncovered a dense and diverse network of microtubule inner proteins (MIPs) that reside within the lumen of doublet microtubules and the central apparatus. These proteins are arranged in a remarkably ordered architecture and contribute to the mechanical stability, periodic organization and functional regulation of the ciliary axoneme. In this Review, we summarize current structural and functional insights into conserved and lineage-specific MIPs, their roles in shaping ciliary architecture, and the consequences of their disruption on ciliary motility and the resulting ciliopathies. We also highlight emerging approaches that are beginning to reveal the specific contributions of MIPs to axonemal integrity, spatial organization and mechanical stability. Together, these advances are reshaping our understanding of how MIPs regulate ciliary structure and function.

The primary cilium is a solitary, microtubule-based organelle present on most vertebrate cells, where it functions as a central hub for sensing and transducing extracellular signals. This Cell Science at a Glance article highlights how primary cilia integrate key signalling pathways - including Hedgehog, G protein-coupled receptor, TRP ion channel, receptor tyrosine kinase and transforming growth factor β superfamily signalling - to regulate cellular processes, tissue architecture and organ function. We also describe how defects in ciliary structure or signalling give rise to ciliopathies, a diverse group of disorders affecting multiple organs and systems. Finally, we explore emerging insights into how dynamic changes in ciliary composition generate cell type- and context-specific signalling signatures, positioning the cilium as a convergence point for multiple signalling branches that coordinate development and homeostasis in time and space. The accompanying poster provides further detail on signalling modules and specializations across cell types.

Cfap298 is a highly conserved gene required for ciliary motility and dynein arm assembly, with known roles in left-right (LR) patterning in zebrafish and links to human ciliopathies. Here, we describe a Cfap298 mutant allele, Cfap298ΔΔS, which selectively disrupts LR axis establishment in mice. Mutant embryos display organ laterality defects and abnormal Nodal, Pitx2 and Lefty1 expression, consistent with an early disruption in LR symmetry breaking. LR asymmetry is established by leftward fluid flow in the node, generated by planar-polarized cilia. Although cfap298 mutations are reported to affect planar polarity, we did not observe changes in cilia position, length or CELSR1 localization within the node, suggesting that Cfap298ΔΔS functions at the level of cilia motility. Accordingly, cilia lining the trachea of Cfap298ΔΔS mutants fail to beat or beat incorrectly. Expression of the Cfap298ΔΔS variant in zebrafish partially rescues body curvature defects but fails to rescue LR defects of cfap298 (kurly) loss-of-function mutants. These results confirm a conserved role for Cfap298 in mammalian LR patterning and identify a previously unreported region of CFAP298 with a conserved and essential role in cilia motility.

Primary cilia are ubiquitous sensory organelles mediating various signaling modalities essential for development and cell homeostasis. Their dysfunction leads to ciliopathies, human disorders often affecting the central nervous system. CEP290 is a major ciliopathy-associated gene that encodes a centrosomal and ciliary transition zone protein. CEP290 has been implicated in different cellular functions, including cell cycle control, ciliogenesis and control of ciliary membrane protein content. To investigate CEP290 dysfunction in human neurons, we generated human induced pluripotent stem cell (iPSC)-derived brain organoids harboring CEP290 mutations. We found that CEP290 deficiency does not affect cell cycle progression or organoid formation, despite a tendency for less mature neuronal populations and formation of choroid plexus in mutant organoids. Expansion microscopy revealed morphologically abnormal ventricular cilia in the CEP290 mutant organoid cells with bulging ciliary membranes around splayed distal axonemal microtubules. Such ciliary abnormalities might represent a tissue-specific consequence revealed by studying a human neuronal organoid model.

The most common genetic cause of the childhood blindness disease Leber congenital amaurosis is mutation of the ciliopathy gene CEP290. Despite extensive study, the photoreceptor-specific roles of CEP290 remain unclear. Using advanced microscopy techniques, we investigated the sub-ciliary localization of CEP290 and its role in mouse photoreceptors during development. CEP290 was found throughout the connecting cilium between the microtubules and membrane, with nine-fold symmetry. In the absence of CEP290 ciliogenesis occurs, but the connecting cilium membrane is aberrant, and sub-structures, such as the ciliary necklace and Y-links, are confined to the proximal connecting cilium. Transition zone (TZ) proteins AHI1 and NPHP1 were abnormally restricted to the proximal connecting cilium in the absence of CEP290, whereas other TZ proteins, like NPHP8 and CEP89 were unaffected. Although outer segment disc formation is inhibited in Cep290 mutant retina, we observed large numbers of extracellular vesicles. These results suggest roles for CEP290 in ciliary membrane structure, outer segment disc formation and photoreceptor-specific spatial distribution of a subset of TZ proteins, which collectively lead to failure of outer segment formation and photoreceptor degeneration.

Living cells of brown algae are difficult to observe in 3D because pigments such as fucoxanthin and chlorophyll diffract light. Furthermore, at the beginning of their life, brown algae develop slowly in seawater. To gain insight into the 3D shape and size of brown algal cells during embryogenesis, we designed a fluorescence probe that labels the plasma membrane efficiently and selectively. Styryl benzoindoleninium sulfonate (SBIS) is a bright orange fluorogenic probe that is soluble and virtually non-emissive in seawater and is activated upon binding to the plasma membrane. Unlike Calcofluor White, SBIS enables observation of cells at thicknesses of up to 25 µm. More importantly, SBIS allows 3D observation of the cells in the growing uniseriate filaments of Ectocarpus sp., the polystichous filaments of Sphacelaria rigidula and the cellular monolayered lamina of Saccharina latissima over periods of up to 7 days. Altogether, these properties allow visualization of entire cell contours in living brown algae, making the study of early development at the cellular level in 4D now possible in these marine organisms.