Scanning electron microscopy of human islet cilia

Polino, Alexander J; Sviben, Sanja; Melena, Isabella; Piston, David W.; Hughes, Jing W.

doi:10.1101/2023.02.15.528685

Cited by 5 publications

(7 citation statements)

References 76 publications

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“…Cilia across spatial scales were tracked by morphology and their identification aided by immunogold labeling of ciliary protein markers. Preservation of ciliary structures was excellent as most cilia axonemes remained intact, exhibiting similar lengths (4-10 µm) and diameters (200-300 nm at base) as previously characterized by us and others 9,11,14,15 . We found moderate magnification (20-35,000x) to give a good overview of the entire ciliary structure.…”

Section: Resultssupporting

confidence: 72%

“…Scanning electron microscopy (SEM) is a high-resolution technique uniquely suited for examining the native 3D conformation of surface structures, an approach that we recently used to characterize the primary cilia axoneme of human islets 9 . In the present study, we combine our SEM protocol with antibody staining to identify specific ciliary protein components.…”

Section: Introductionmentioning

confidence: 99%

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Immuno-Scanning Electron Microscopy of Islet Primary Cilia

Sviben,

Polino,

Melena

et al. 2024

Preprint

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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 contain 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 immuno-labeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the 3D 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, immuno-labeling, and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

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Section: Resultssupporting

confidence: 72%

Section: Introductionmentioning

confidence: 99%

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

Sviben,

Polino,

Melena

et al. 2024

Preprint

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“…Potentially, the axoneme might be already sinistrally twisted in the absence of dynein forces. Interestingly, structural sinistral twist was reported for nonmotile 9+0 axonemes of human islet cilia 43 . Sinistral structural components were also reported for motile 9+2 axonemes, such as the central apparatus 23,44 , or the heads of the radial spokes 45 .…”

Section: Relation To Past Workmentioning

confidence: 98%

Twist - torsion coupling in beating axonemes

Striegler,

Friedrich,

Diez

et al. 2024

Preprint

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Motile cilia and flagella are ubiquitous cell appendages whose regular bending waves pump fluids across tissue surfaces and enable single-cell navigation. Key to these functions are their non-planar waveforms with characteristic torsion. It is not known how torsion, a purely geometric property of the shape, is related to mechanical deformations of the axoneme, the conserved cytoskeletal core of cilia and flagella. Here, we assess torsion and twist in reactivated axonemes isolated from the green alga Chlamydomonas reinhardtii. Using defocused darkfield microscopy and beat-cycle averaging, we resolve the 3D shapes of the axonemal waveform with nanometer precision at millisecond timescales. Our measurements reveal regular hetero-chiral torsion waves propagating base to tip with a peak-to-peak amplitude of 22 °/μm. To investigate if the observed torsion results from axonemal twist, we attach gold nanoparticles to axonemes to measure its cross-section rotation during beating. We find that locally, the axonemal cross-section co-rotates with the bending plane. This co-rotation presents the first experimental evidence for twist-torsion coupling and indicates that twist waves propagate along the axoneme during beating. Our work thus links shape to mechanical deformation of beating axonemes, informing models of motor regulation that shape the beat of motile cilia.

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“…Complementary to the quantitative data of UExM, EM of beta cell primary cilia deep within isolated islets and pancreas tissue allowed us to assess their ultrastructural features in situ. Previous SEM imaging (28) and our own SEM data of intact isolated islets only allow for imaging their peripheral surfaces. In such cases the cilia appeared as structures extending into an empty extracellular space (Fig.…”

Section: Primary Cilia Are Spatially Restricted Within Long Ciliary P...mentioning

confidence: 99%

“…Three-dimensional (3D) reconstructions of the axoneme of beta cell primary cilia are currently not available. Until now the ultrastructure of beta cell primary cilia and their axonemes has been investigated only by two-dimensional (2D) transmission electron microscopy (TEM) (34; 33; 22; 24) as well as scanning electron microscopy (SEM) (28). Some of these studies indicate a dis-organization of the axoneme.…”

Section: Introductionmentioning

confidence: 99%

Structure, interaction, and nervous connectivity of beta cell primary cilia

Müller,

Klena,

Pang

et al. 2023

Preprint

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Primary cilia are sensory organelles present in many cell types. Based on an array of microtubules termed axoneme they form a specialized membrane compartment partaking in various signaling processes. Primary cilia of pancreatic islet beta cells play a role in autocrine and paracrine signaling and are linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of complete mouse and human beta cell cilia, revealing a disorganized 9+0 axoneme structure. Within the islet, cilia are spatially confined within deep ciliary pockets or squeezed into narrow extracellular spaces between adjacent cells. Beta and alpha cell cilia physically interact with neighboring islet cells pushing and strongly bending their plasma membranes. Furthermore, beta cells can contain multiple cilia that can meet with other islet cell cilia in the extracellular space. Additionally, beta cell cilia establish connections with islet-projecting nerves. These findings highlight the pivotal role of beta cell primary cilia in islet cell connectivity, pointing at their potential functional role in integrating islet intrinsic and extrinsic signals. These novel insights contribute to understanding their significance in health and diabetes.

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Scanning electron microscopy of human islet cilia

Cited by 5 publications

References 76 publications

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

Twist - torsion coupling in beating axonemes

Structure, interaction, and nervous connectivity of beta cell primary cilia

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