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Significance The primary cilium is a small subcompartment of the cell but has powerful influence on pancreatic islet function. In this study, we find a critical role for cilia in regulating β-cell function and energy metabolism. Importantly, the deletion of β-cell cilia disrupts intercellular communication and leads to islet dysfunction and diabetes, as seen in a number of human ciliopathy syndromes. These results should help elucidate pathophysiology of human ciliopathy and aid the development of pharmacologic agents targeting primary cilia that may lead to a more effective treatment for human diabetes., Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking β-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the β-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of β-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate β-cell–intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes.

We describe here a protocol for performing simultaneous spatiotemporal computational super-resolution and multi-parametric fluorescence microscopy. Our approach does not need specialized instrumentation, utilizes a GPU for faster data evaluation, and produces mutually consistent structure and dynamics data.

We describe a protocol for the preparation of live cell samples for fluorescence spectroscopy and computational super-resolution imaging. We detail here how to culture, transfect, and prepare the cells for fluorescence applications.

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements., Super-resolution microscopy and single molecule fluorescence spectroscopy require optimisation of the temporal or spatial resolution, which are usually mutually exclusive. Here the authors report a GPU-supported, camera-based strategy to achieve high spatial and temporal resolution from the same dataset.

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~60 nm), temporal dynamics (≤ 2 ms), and molecular brightness from the exact same data set. We demonstrate the applicability and advantages of multi-parametric measurements to monitor the super-resolved structure and dynamics of two different biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin network. Multi-parametric analysis suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides new biological knowledge that cannot be obtained from sequential measurements.

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.

Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live-cell environments.

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

There has been increasing interest in biophysical studies on live organisms to gain better insights into physiologically relevant biological events at the molecular level. Zebrafish (Danio rerio) is a viable vertebrate model to study such events due to its genetic and evolutionary similarities to humans, amenability to less invasive fluorescence techniques owing to its transparency and well-characterized genetic manipulation techniques. Fluorescence techniques used to probe biomolecular dynamics and interactions of molecules in live zebrafish embryos are therefore highly sought-after to bridge molecular and developmental events. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are two robust techniques that provide molecular level information on dynamics and interactions respectively. Here, we detail the steps for applying confocal FCS and FCCS, in particular single-wavelength FCCS (SW-FCCS), in live zebrafish embryos, beginning with sample preparation, instrumentation, calibration, and measurements on the FCS/FCCS instrument and ending with data analysis.

Wnt3 is a signaling protein involved in development and disease (oncogenesis and tetraamelia syndrome). Recently, we have shown that Wnt3 regulates cerebellum development using zebrafish Wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP (Teh et al., Development 2015). We identified several fractions of Wnt3: i) an intracellular and ii) a secreted fraction, which comprises of monomeric Wnt3 as well as its complexes, and, iii) a membrane-bound fraction. The membrane-bound O-acyltransferase Porcupine is required for Wnt palmitoylation, secretion and biological activity. It was shown in vitro that for membrane localization in Wnt3-expressing cells Wnt3 undergoes two lipid modifications by palmitoylation at C77 and S209. Such posttranslational modifications have been linked with protein association with cholesterol-dependent lipid domains. This raises the question whether Wnt3 is targeted to these domains in live zebrafish. We therefore used SPIM-FCS and the FCS diffusion laws, which characterize the dependence of the diffusion coefficient on the observed area to deduce deviations from free diffusion, to characterize Wnt3 distribution on plasma membranes in zebrafish embryos.First, we demonstrate that the diffusion laws can be applied in a 3D environment of SPIM-FCS by characterizing lipid distributions in giant unilamellar vesicles (GUVs) of various compositions. Second, we applied this methodology to analyze Wnt3 distribution on plasma membranes in live Wnt3 transgenic embryos. This demonstrated that in vivo the Wnt3 membrane-bound fraction also shows consistent raft partitioning. Third, upon reduction of Wnt3 secretion by a specific inhibitor of Porcupine, Wnt3 partitioning into lipid domains is also reduced. Taken together this demonstrates that in developing zebrafish embryos the plasma membrane fraction of Wnt3 to be secreted needs to be compartmentalized into specialized lipid domains.