Giulia Ossato

A Two-Step Path to Inclusion Formation of Huntingtin Peptides Revealed by Number and Brightness Analysis

Protein aggregation is a hallmark of several neurodegenerative diseases including Huntingtons disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A-I and high density lipoproteins.

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.

Bimetallic nanopetals for thousand-fold fluorescence enhancements

We present a simple, ultra-rapid and robust method to create sharp nanostructures—nanopetals—in a shape memory polymer substrate demonstrating unprecedented enhancements for surface enhanced sensing over large surface areas. These bimetallic nanostructures demonstrate extremely strong surface plasmon resonance effects due to the high density multifaceted petal structures that increase the probability of forming nanogaps. We demonstrate that our nanopetals exhibit extremely strong surface plasmons, confining the emission and enhancing the fluorescence intensity of the nearby high-quantum yield fluorescein by >4000×. The enhancements are confined to the extremely small volumes at the nanopetal borders. This enables us to achieve single molecule detection at relatively high and physiological concentrations.

Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

Golgi sorting regulates organization and activity of GPI proteins at apical membranes

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

Mapping Retinoids in Live P19 Cells with Autofluorescence Phasorflim Imaging

Recently a label-free FLIM imaging method has been proposed to distinguish metabolic states of germ cells (Stringari et al, 2011). This method, based on the phasor analysis of autofluorescence lifetime (AF-phasorFLIM), avoids some problems of state-of-art approaches due to multi-exponential fitting. The capability of phasorFLIM analysis to separate the different molecular species in a pixel lies on the rule of phasor addition. PhasorFLIM identifies molecules by their position in the phasor plot because every molecular species has a specific phasor that can be calibrated in reference samples.We applied phasorFLIM to detect retinoids autofluorescence in live P19 embryonal carcinoma cells that undergo differentiation when treated with retinoic acid (RA).RA and retinol are essential for cell-cell signaling during vertebrate organogenesis. RA is involved in stem cell differentiation and it is a morphogen. It is a diffusible signal that generates patterns in embryo because it induces different cellular responses depending on its local concentration. The presence of RA in tissues has been inferred through gene activation, and the RA average tissular concentration measured by HPLC. However, the spatial distribution in tissues and cells of the active molecule has never been observed or quantified directly.We built a database of phasorFLIM standards of the major molecules that contribute to autofluorescence. The database allows assigning the contribution of fluorescent molecules to P19 autofluorescence.P19 cells were treated with retinol to follow in time the effect of the molecule on cellular metabolism. PhasorFLIM analysis was performed using SimFCS and showed an increase of retinoids fluorescence after treatment. In cells treated with retinol, we observed retinol in plasma and nuclear membrane and an increase of RA in the nucleus.AF-phasorFLIM allowed determining, distinguishing and localizing the contribution of retinoids to autofluorescence in live P19 cells.