Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Brody, Steven L.; Pan, Jiehong; Huang, Tao; Xu, Jian; Xu, Huihui; Koenitizer, Jeffrey; Brennan, Steven K.; Nanjundappa, Rashmi; Saba, Thomas G.; Berical, Andrew; Hawkins, Finn J.; Wang, Xiangli; Zhang, Rui; Mahjoub, Moe R.; Horani, Amjad; Dutcher, Susan K.

doi:10.1101/2024.03.20.585965

Cited by 4 publications

(1 citation statement)

References 115 publications

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“…We did not observe discrete IFT train-like structures corresponding to IFT88 immunolabeling on the axonemal surface and reason that the train complexes might have been partially disrupted by the demembranation process. Other components of the axoneme might also have been disrupted, although the core microtubule bundle remained intact and we did not observe microtubule splaying as sometimes can be seen with EM specimen damage or structural mutations affecting ciliary microtubular integrity ( Brody et al, 2024 preprint; Polino et al, 2023 ).…”

Section: Resultsmentioning

confidence: 52%

Immuno-scanning electron microscopy of islet primary cilia

Sviben,

Polino,

Melena

et al. 2024

Journal of Cell Science

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The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

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Section: Resultsmentioning

confidence: 52%

Immuno-scanning electron microscopy of islet primary cilia

Sviben,

Polino,

Melena

et al. 2024

Journal of Cell Science

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Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia

Abou Alaiwa,

Hilkin,

Price

et al. 2024

Preprint

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Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

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Cilia dysfunction in the lateral ventricles after neonatal intraventricular hemorrhage does not lead to functional changes in cilia-based CSF flow networks

Pan,

Ramagiri,

Yang

et al. 2024

Preprint

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Intraventricular hemorrhage (IVH) has long been thought to lead to motile cilia dysfunction whereby intraventricular blood breakdown products damage and slough cilia from the ependymal wall. However, specifically how IVH may affect cilia development, structure, and transcriptional activation is not well-understood. Moreover, the impact of blood breakdown product-mediated cilia damage on the functional organization of cilia-based CSF flow networks is unknown. Here, we show hemoglobin exposure affects the number of ciliated ependymal cells in the lateral ventricle (LV) but does not impact in vitro beat frequency of the remaining cilia. Ultrastructurally, IVH decreases the total number of ciliary tufts without impacting axoneme structure. IVH does not result in changes in the expression of cilia-related genes and instead leads to downregulation of neurogenesis markers in parallel with innate immune upregulation. Functionally, we identify three previously uncharacterized cilia-mediated CSF flow domains in the LV lateral wall and show that IVH does not result in widespread disruption of their functional organization. These data de-emphasize cilia as a major contributor to global CSF dysfunction after IVH, and instead call attention to preserving the neurodevelopmental environment and preventing runaway innate immune system activation, as considerations to developing treatment strategies to prevent posthemorrhagic hydrocephalus and other neurodevelopmental sequelae.

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Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Cited by 4 publications

References 115 publications

Immuno-scanning electron microscopy of islet primary cilia

Immuno-scanning electron microscopy of islet primary cilia

Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia

Cilia dysfunction in the lateral ventricles after neonatal intraventricular hemorrhage does not lead to functional changes in cilia-based CSF flow networks

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