Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeleton.

Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic ß cells, a long-standing model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating ß cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhance glucose-stimulated insulin secretion. Using scanning electron microscopy, we directly visualize the cortical cytoskeleton, allowing us to validate the effect of these filament-disrupting chemicals. We find the state of the cortical actin layer does not correlate with levels of insulin secretion, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton.

Fabrication of a multifaceted mapping mirror using two-photon polymerization for a snapshot image mapping spectrometer.

A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93 nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12 nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.

Palmitoylation couples insulin hypersecretion with ß cell failure in diabetes.

Hyperinsulinemia often precedes type 2 diabetes. Palmitoylation, implicated in exocytosis, is reversed by acyl-protein thioesterase 1 (APT1). APT1 biology was altered in pancreatic islets from humans with type 2 diabetes, and APT1 knockdown in nondiabetic islets caused insulin hypersecretion. APT1 knockout mice had islet autonomous increased glucose-stimulated insulin secretion that was associated with prolonged insulin granule fusion. Using palmitoylation proteomics, we identified Scamp1 as an APT1 substrate that localized to insulin secretory granules. Scamp1 knockdown caused insulin hypersecretion. Expression of a mutated Scamp1 incapable of being palmitoylated in APT1-deficient cells rescued insulin hypersecretion and nutrient-induced apoptosis. High-fat-fed islet-specific APT1-knockout mice and global APT1-deficient db/db mice showed increased ß cell failure. These findings suggest that APT1 is regulated in human islets and that APT1 deficiency causes insulin hypersecretion leading to ß cell failure, modeling the evolution of some forms of human type 2 diabetes.

Conformational plasticity of NaK2K and TREK2 potassium channel selectivity filters.

The K+ channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K+ binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K+-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K+ channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K+ conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K+ channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K+ channels. Our results establish the generality of K+-induced SF conformational stability across the K+ channel superfamily, and introduce an approach to study manipulation of channel selectivity.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy.

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.

Cytoskeleton-dependent clustering of membrane-bound prion protein on the cell surface.

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.

Primary cilia control glucose homeostasis via islet paracrine interactions.

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 ß-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the ß-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 ß-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 ß-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.

Fluorescence Correlation and Cross-Correlation Spectroscopy in Zebrafish.

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.

The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study.

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.

Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons.

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.

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes.

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7 × 7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Characterization of Lipid and Cell Membrane Organization by the Fluorescence Correlation Spectroscopy Diffusion Law.

The plasma membrane organization of live cells defines a plethora of cellular processes important for cell functionality. Many membrane structures that define this organization exist at a spatial resolution below the optical diffraction limit and are highly dynamic. Therefore, a method with millisecond time resolution and nanometer spatial resolution is required for the investigation of plasma membrane organization. However, spatial and temporal resolutions of the currently available biophysical techniques are often mutually exclusive. In a novel realization, Lenne and coworkers developed a spot-variation modality of fluorescence correlation spectroscopy (FCS), also known as FCS diffusion law, to harvest nanoscopic information from microscopic measurements. The FCS diffusion law, so far, has been instrumental to decode the physico-chemical origin of membrane organization and its relationship with biological processes. Overall, the structural information of plasma membrane obtained by FCS diffusion law provides a better understanding of its coupling to the underlying cellular processes.