Structural characterization of primary cilia using accelerated piezoelectrically driven STED nanoscopy
Creators: Nathwani, Bhavik Bharat
Publisher: name: Columbia University
Year: 2012
Subjects: Cytology

description: Primary cilia are non-motile, hair-like projections occurring on most mammalian cell types. They play essential roles in transduction of chemical and mechanical signals across the cell membrane. For example, primary cilia are able to transduce sonic hedgehog signals, necessary in embryonic development and adult stem cell functions. Recent work on primary cilia has demonstrated correlations between primary cilia morphology and its ability to sense/transduce signals. Several such studies have underscored the need for detailed study of morphology of primary cilia and structure-function mapping of its morphology with its ability to transduce signals. However, the size scale of the primary cilium makes it very challenging to extract biologically relevant morphometric features using conventional imaging techniques. The molecular architecture of the primary cilium is beyond the resolvability of conventional diffraction limited optical imaging techniques. Data from non-optical tools such as electron microscopy have been limited by the need for dehydration during sample prep. Advent of superresolution optical imaging approaches has only recently made it possible to probe primary cilia morphologically to study its structure in physiologically interesting environments.  Signaling pathways regulated by primary cilia are critical to embryo development and organogenesis. Therefore, it would be interesting to study primary cilia both in somatic (adult) cells while simultaneously comparing and contrasting it with their occurrence on stem cells. Human induced pluripotent stem cell (hiPSC) reprogramming possesses enormous potential in stem cell research and disease modeling. Chemical and mechanical signaling has been implicated in maintenance of pluripotency of hiPSCs and their differentiation pathways toward various lineages, where primary cilia have been shown to play a critical role in mechano-chemical signaling across a wide spectrum of cell types. The functions of primary cilia in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. Therefore, in order to study primary cilia morphology on both somatic cells as well as hiPSCs, we developed a superresolution nanoscopy system using the stimulated emission depletion (STED) technique with novel accelerated piezoelectric control (apSTED). This improved STED system achieved a reduction in photobleaching rates from ~80% to ~10% while maintaining superresolution, ~50 nm at the focal plane for biological samples. Subsequently, we focused on conducting comparative morphometric studies of primary cilia found on somatic cells and hiPSCs. Our work was the first to systematically demonstrate the existence of primary cilia on hiPSCs. Using quantitative PCR assays, we demonstrated high levels of expression of primary cilia signaling partners, such as Patched1, Smoothened, and members of Gli family. Comparative morphometric analysis revealed that the mean length of reprogrammed cells was shorter than those of parental human fibroblasts. Morphometric analyses revealed that reprogramming resulted in an increase in curvature of primary cilia from ~0.015 µm to 0.064 µm, indicating an underlying ~4-fold decrease in their rigidity, and a decrease in length of primary cilia from ~2.38 µm to ~1.45 µm. Furthermore, reprogramming resulted in fewer primary cilia displaying either kinked or punctated geometries. Custom-built software scripts were developed to extract and analyze superresolution apSTED imaging data collected on fibroblast primary cilia. Using apSTED, we were able to measure local variations in primary cilia curvature. A review of confocal data revealed that such variations in curvature were either completely missed or were significantly underestimated. We also utilized our technique to study macromolecular complexes within transition zone; a structure found at the base of primary cilia that plays a significant role in ciliogenesis and in maintaining structural integrity of primary cilia. Our data provides the first visualization of two important transition zone members, Tctn-2 and Cep290. We were able to demonstrate structural detail heretofore impenetrable to conventional imaging techniques. Furthermore, quantification of spatial distribution of these molecules, ~160 nm for Tctn-2 and ~180 nm for Cep290, provides evidence to indicate the relative positioning of these molecules within the transition zone. These studies highlight the advantages of using apSTED to study primary cilia and provide tools that could enable the deciphering of the architecture of the transition zone in primary cilia.