Structural characterization of primary cilia using accelerated piezoelectrically driven STED nanoscopy

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

Developing luminescent nanoprobes for labeling focal adhesion complex proteins and performing combined AFM-TIRF imaging of these conjugates

Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and made biocompatible. A process to coat the QDs with silk fibroin, a fibrous protein derived from the Bombyx mori silk worm, is described. Following the coating process, the characterization of size, optical properties and biocompatibility profile of these particle systems is described. In addition, conjugation of the silk fibroin coated QDs to different labeling proteins such as phalloidin and streptavidin is described. Proteins on the surface of ovarian cancer cells (HeyA8) and of cytoskeletal components participating in the formation of focal adhesion complex (FAC), such as F-actin in endothelial cells (HUVECS) were labeled using the bio-conjugated QDs. Various imaging techniques such as epi-fluorescence, TIRF and AFM were used to study the QD labeled cells. Overall the project has produced luminescent nanoprobes that enable the study of FAC formation dynamics and potentially a better in vivo fluorescent marker tool

Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of similar to 90nm diameter and similar to 250nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of similar to 8nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology

A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion

Soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications