Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration.

The use of super-resolution microscopy in recent years has revealed that proteins often form small assemblies inside cells and are organized in nanoclusters. However, determining the copy number of proteins within these nanoclusters constitutes a major challenge because of unknown labeling stoichiometries and complex fluorophore photophysics. We previously developed a DNA-origami-based calibration approach to extract protein copy number from super-resolution images. However, the applicability of this approach is limited by the fact that the calibration is dependent on the specific labeling and imaging conditions used in each experiment. Hence, the calibration must be repeated for each experimental condition, which is a formidable task. Here, using cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample, we demonstrate that the DNA-origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any green-fluorescent-protein-fused protein of interest. To do so, we first quantified the copy number of dynein motors within nanoclusters in the cytosol and along the microtubules. Interestingly, this quantification showed that dynein motors form assemblies consisting of more than one motor, especially along microtubules. This quantification enabled us to use the HeLa IC74 cells as a reference sample to calibrate and quantify protein copy number independently of labeling and imaging conditions, dramatically improving the versatility and applicability of our approach.

PEGylated gold nanorods as optical trackers for biomedical applications: an in vivo and in vitro comparative study.

Gold nanorods (AuNRs) are eligible for a variety of biological applications including cell imaging, sensing, and photothermal therapy thanks to their optical properties. The aim of this work is to show how AuNRs could be employed as non-photobleachable optical contrast agents for biomedical applications. In order to demonstrate the feasibility of their use as optical trackers, we employed two-photon emission confocal microscopy on cells incubated with PEGylated AuNRs. Remarkably, AuNRs were localized mostly in the perinuclear zone and microscopy characterization showed the presence of a considerable number of rods inside cell nuclei. Furthermore, we estimated the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs as a function of administered dose on HeLa/3T3 cell lines and on zebrafish during development, employed as an in vivo model. Eventually, we observed good agreement between in vivo and in vitro experiments. The employed AuNRs were prepared through a photochemical protocol here improved by tuning the amount of the cationic surfactant cetyltrimethylammonium bromide for the achievement of AuNRs at two different aspect ratios. Furthermore we also investigated if the AuNR aspect ratio influenced the toxicity and the efficiency of cellular uptake of the PEGylated AuNRs in HeLa/3T3 cell lines and in zebrafish embryos.

Targeting the Gut: A Systematic Review of Specific Drug Nanocarriers.

The intestine is essential for the modulation of nutrient absorption and the removal of waste. Gut pathologies, such as cancer, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and celiac disease, which extensively impact gut functions, are thus critical for human health. Targeted drug delivery is essential to tackle these diseases, improve therapy efficacy, and minimize side effects. Recent strategies have taken advantage of both active and passive nanocarriers, which are designed to protect the drug until it reaches the correct delivery site and to modulate drug release via the use of different physical-chemical strategies. In this systematic review, we present a literature overview of the different nanocarriers used for drug delivery in a set of chronic intestinal pathologies, highlighting the rationale behind the controlled release of intestinal therapies. The overall aim is to provide the reader with useful information on the current approaches for gut targeting in novel therapeutic strategies.

Patient's dermal fibroblasts as disease markers for visceral myopathy.

Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research.

Nanotechnology applications in medicine.

In recent years there has been a rapid increase in nanotechnology applications to medicine in order to prevent and treat diseases in the human body. The established and future applications have the potential to dramatically change medical science. The present paper will give a few examples that could transform common medical procedures.

Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy.

In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in bovine rod outer segments.

PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.

GABAB receptor intracellular trafficking after internalization in Paramecium.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

Dimethyl-pepep: a DNA probe in two-photon excitation cellular imaging.

Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.

Nanocapsules: coating for living cells.

One of the most promising tools for future applications in science and medicine is the use of nanotechnologies. Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner. The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response. With these shells, material can be delivered to predefined organs. In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules. Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy. The measurements reveal an increased surface charge in the region of buds developed prior encapsulation. In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated. The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles. The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.

Patch-clamp recordings in isolated sponge cells (Axinella polypoides).

Sponges are the most ancient known metazoans. Their cells are specialised but not organised into tissues or organs. Recordings of action potential-like propagating electrical impulses suggested that electrical signalling may occur between sponge cells, but the characterization of ionic channels in these cells is still at the beginning. Actually, sponge cell surfaces are covered by a complex glycocalyx and long-chain fatty acids are present in the lipid core of their membranes. In these experimental conditions, a low percentage of tight seals (3%) was obtained applying the patch-clamp technique to cells isolated from the Mediterranean Demospongia Axinella polypoides. This paper shows in detail how difficulties can be overcome making use of trivalent cations in the extracellular solution and how electrophysiological measurements can be performed on sponge cell membranes. A potassium selective conductance is shown as an example. We suggest that the presented methodology could also be applied to other cell types.

Protection capabilities of nanostructured shells toward cell encapsulation: a Saccharomyces/Paramecium model.

In this work we report the formation of nanostructured hybrid objects, made up of living cells encapsulated in a protective multilayer shell assembly of nanostructured polyelectrolyte. Such constructs can be hosted on nanostructured surfaces or can be installed around living organisms, at the right time. Their construction is based on the layer-by-layer (LbL) self-assembly of two oppositely alternated charged polyelectrolytes (PEs) on cell membranes as earlier done for nanocapsules or fuzzy structured nanoshells. This communication reports the optimal conditions for cell encapsulation in terms of nanoshell design and construction.

Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling.

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

3D localized photoactivation of pa-GFP in living cells using two-photon interactions.

We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. The aim in this work was to evaluate the use of two-photon interactions for confining the molecular switching of pa-GFP in the bright state. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation and two-photon mode demonstrates clearly the better three- dimensional (3D) confinement and the possibility of selection of cell volumes of interest. This enables molecular trafficking studies at high signal to noise ratio.

Encapsulated living cells on microstructured surfaces.

The immobilization of cells in defined arrays (cell patterning) is a key step towards cell-based biosensors or other cell-based devices. While cell patterning is usually achieved by modifying the surface on which only the cells should adhere and leaving the cells unmodified, we present here a different approach in which cells are first coated with polyelectrolytes and subsequently immobilized on patterned surfaces. By coating, the cells are protected and their interactions with the substrate are modified such that patterning is simplified. We used microcontact printing of polyelectrolytes to structure surfaces such that regions of opposite charges and the same charge as the cell coating were present and found that we can thus achieve patterning of the coated yeast cells. In accordance with prior work, we find that coating does not kill the cells and coated GFP-expressing cells still function after immobilization, which we checked by fluorescence microscopy.

ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges.

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel, with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae). Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress. This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.