Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs)
Abstract
The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real-time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 3 or 4 cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explant in real-time. Furthermore, we find that this methodology for direct cell type specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of secretory and goblet cell associated antigen passages (SAPs and GAPs) enable luminal antigen sampling. Given that airway secretory cells can be stimulated to make mucous within hours, we suspect that both SAPs and GAPs are also used for luminal antigen sampling in the airway. This hypothesis is supported by our observation that secretory cells with airway SAPs are frequently juxtaposed to antigen presenting cells.
Data availability
All data is included in the manuscript and generated custom scripts are included on https://github.com/vss11/Label-free-autofluorescence.
Article and author information
Author details
Funding
Cystic Fibrosis Foundation (003338L121)
- Viral S Shah
NHLBI Division of Intramural Research (5R01HL142559)
- Charles P Lin
- Jayaraj Rajagopal
NHLBI Division of Intramural Research (RO1HL118185)
- Jayaraj Rajagopal
NHLBI Division of Intramural Research (1R01HL157221)
- Jayaraj Rajagopal
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Mice were maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at the Massachusetts General Hospital (MGH). All procedures were performed with Institutional Animal Care and Use Committee (IACUC)-approved protocols (#2009N000119). MGH is accredited by AAALAC International, has an assurance with the Office of Laboratory Animal Welfare (OLAW) and is registered with the United States Department of Agriculture (USDA). Euthanasia was performed via house line CO2-mediated asphyxiation and confirmatory cervical dislocation consistent with the recommendations of the American Veterinary Medical Association.
Copyright
© 2023, Shah et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,822
- views
-
- 215
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
-
- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.