Number and brightness analysis reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1 in live cells.

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

Application limits and data correction in number of molecules and brightness analysis.

Number of molecules and Brightness (N&B) has been proposed for measuring the molecular brightness and number of fluorophores in time-sequence of images, in live cells. If the fluorescently tagged-proteins are mobile in the illumination volume, the stoichiometry of their oligomers can be derived from the increase of the brightness of the fluorescent dyes due to clustering. We examine aspects concerning extra-fluctuation effects induced by cell shifts and photobleaching, which yield large overestimates of the clusters size and sub-unit counts. We develop an offline corrective approach consisting in frame re-alignment and boxcar filtering for recovering precision of the analysis. Using simulations we derive general criteria for approaching this analysis, and assess the application limits of the corrective procedure. We tested the approach in extreme experimental conditions (few pixels, large extra-variance perturbations), in which we analyzed the minimal increases of brightness as that expected between a monomeric and dimeric GPI-mEGFP constructs. We show how most of the perturbing effects can be abolished, and obtain the correct the brightness of GPI-mEGFP monomers and dimers.

Proteomic identification of aldolase A as an autoantibody target in patients with atypical movement disorders.

We tried to identify the target/s of autoantibodies to basal ganglia neurons found in a patient with hyperkinetic movement disorders (HMD) characterized by rapid, rhythmic involuntary movements or spasms in both face and neck. Patient and control sera were used in Western blot to probe mouse brain homogenates. Two-dimensional gel electrophoresis (2-DE) SDS-PAGE protein spots recognized by the patient's antibodies were excised and sequenced by mass spectrometry analysis, and the glycolytic enzyme aldolase A was identified as the antigen recognized by the patient's autoantibodies. To assess relevance and specificity of these antibodies to the identified targets as biomarkers of autoimmunity in movement disorders, autoantibody responses to the identified target were then measured by ELISA in various diseases of the central nervous system. Anti-aldolase A autoantibodies were associated mainly with HMD (7/17, 41%) and Parkinson's disease (4/30, 13%) patients, and undetectable in subjects with other inflammatory and non-inflammatory central nervous system diseases. We, thus, identified aldolase A as an autoantigen in a sub-group of patients with HMD, a clinically ill-defined syndrome. Anti-aldolase A antibodies may represent a useful biomarker of autoimmunity in HMD patients.

Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane.

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.

Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins.

BACKGROUND: All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture. RESULTS: To obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles. CONCLUSION: Because reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.

Evaluation of the molecular mechanisms involved in the gain of function of a Li-Fraumeni TP53 mutation.

The TP53 tumor suppressor gene is the most frequent target for genetic alterations in human cancer. TP53 gene alterations may result in the gain of oncogenic functions such as neoangiogenesis and resistance to therapy. The TP53 germ line mutation c.659A>C (p.Y220S) was identified in stored DNA from related patients with Li-Fraumeni syndrome (LFS) who died after developing clinically aggressive tumors. All of the patients were treated with protocols that included doxorubicin hydrochloride (DX) as a pivotal drug. To define the in vitro mutational phenotype of this germ line mutation, we used murine fibroblasts explanted from wild-type (wt) and p53 knockout (KO) mice from the same littermate. p53Y220S and p53R175H fibroblasts, obtained from p53KO fibroblasts transfected with expression vectors encoding the human Y220S and R175H p53 mutants, respectively, exhibited resistance to DX treatment. Moreover, p53Y220S fibroblasts exhibited angiogenetic properties, and after DX treatment, p53Y220S failed to translocate into the nucleus and showed an increase in its cytosolic levels. DX treatment does not influence p53 distribution within the nuclear and cytosolic compartments in p53R175H fibroblasts. Peroxiredoxin II (Prx II), a protein that is involved in eliminating reactive oxygen species (ROS), showed increased expression intensity in p53Y220S fibroblasts after DX treatment, as observed by two-dimensional electrophoresis analysis. Moreover, Thioredoxin (Trx), a protein that cooperates with Prx II, is overexpressed in p53Y220S mutants under basal conditions. These data suggest a relationship between the presence of the p53Y220S mutation and enhanced levels of Prx II and Trx in mutant fibroblasts. Since one of the mechanisms of the DX antitumor effect has been ascribed to production of ROS, future studies will evaluate the involvement of PrxII and Trx in the chemoresistance of p53Y220S fibroblasts to DX.

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening.

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.