Biomedical subjects

Mario Faretta

Publications and source records attributed to Mario Faretta.

12 recordsLinked to original sources

PubMed2006

Multi-photon excitation microscopy.

Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Animals↗

PubMed2006

In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.

BACKGROUND: Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. RESULTS: To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31(comet), a spindle-assembly checkpoint inhibitor. CONCLUSIONS: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.

Calcium-Binding Proteins↗

PubMed2005

Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region.

This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at lambda approximately 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at lambda = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.

Fluorescent Dyes↗

PubMed2005

The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint.

BACKGROUND: The spindle assembly checkpoint (SAC) imparts fidelity to chromosome segregation by delaying anaphase until all sister chromatid pairs have become bipolarly attached. Mad2 is a component of the SAC effector complex that sequesters Cdc20 to halt anaphase. In prometaphase, Mad2 is recruited to kinetochores with the help of Mad1, and it is activated to bind Cdc20. These events are linked to the existence of two distinct conformers of Mad2: a closed conformer bound to its kinetochore receptor Mad1 or its target in the checkpoint Cdc20 and an open conformer unbound to these ligands. RESULTS: We investigated the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to Cdc20. We report that a closed conformer of Mad2 constitutively bound to Mad1, rather than Mad1 itself, is the kinetochore receptor for cytosolic open Mad2 and show that the interaction of open and closed Mad2 conformers is essential to sustain the SAC. CONCLUSIONS: We propose that closed Mad2 bound to Mad1 represents a template for the conversion of open Mad2 into closed Mad2 bound to Cdc20. This simple model, which we have named the "Mad2 template" model, predicts a mechanism for cytosolic propagation of the spindle checkpoint signal away from kinetochores.

Calcium-Binding Proteins↗

PubMed2005

New method to detect histone acetylation levels by flow cytometry.

BACKGROUND: Reversible histone acetylation affects chromatin structural organization, thus regulating gene expression and other nuclear events. Levels of histone acetylation are tightly modulated in normal cells, and alterations of their regulating mechanisms have been shown to be involved in tumorigenesis. METHODS: We developed a new flow cytometric technique for detection of histone acetylation, based on a specific monoclonal antibody that recognizes acetylated histone tails. Bivariate analysis for histone acetylation levels and DNA were performed to study modulation of chromatin organization during the cell cycle and after induction of histone hyperacetylation by the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Histone acetylation and transcription levels were monitored during differentiation induced by retinoic acid alone or in combination with TSA. Blood samples from patients were analyzed with the described protocol to monitor the effects of HDAC inhibitors in vivo and validate the developed protocol for clinical usage. RESULTS: Flow cytometric detection of acetylation status can successfully detect modifications induced by HDAC inhibitor treatment in vivo as demonstrated by analysis of various blood samples from patients treated with valproic acid. Changes in acetylation levels during the cell cycle demonstrated a reproducible increase in histone acetylation during the replication phase that was subsequently decreased at the G2M entrance, thus paralleling the behavior of DNA replication and transcriptional activity. CONCLUSIONS: Multiparameter analysis of histone acetylation and expression of molecular markers, DNA ploidy, and/or cell cycle kinetics can provide a quick and statistically reliable tool for the diagnosis and evaluation of treatment efficacy in clinical trials using HDAC inhibitors.

Acetylation↗

PubMed2004

From cells to tissues: fluorescence confocal microscopy in the study of histological samples.

Our knowledge of the genetic mechanisms controlling cell proliferation and differentiation usually originates from in vitro cultured cell line models. However, the definition of the molecular switches involved in control of homeostasis and the understanding of the changes occurring in neoplastic transformation require looking at single cells as the components of a complex tissue network. Histological examination of tissue samples can gain a substantial amount of information from high-resolution fluorescence analysis. In particular, confocal microscopy can help in the definition of functional pathways using multiparameter analysis. In this report, we present acquisition and analysis procedures to obtain high-resolution data from tissue sections. Confocal microscopy coupled to computational restoration, statistical evaluation of spatial correlations, and morphological analysis over large tissue areas were applied to colorectal samples providing a molecular fingerprint of the biological differences inferred from classical histological examination.

Animals↗

PubMed2004

Sgt1 is required for human kinetochore assembly.

Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.

Calcium-Binding Proteins↗

PubMed2002

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence.

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

Animals↗

PubMed2002

A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins.

Ubiquitination is a post-translation modification in which ubiquitin chains or single ubiquitin molecules are appended to target proteins, giving rise to poly- or monoubiquitination, respectively. Polyubiquitination targets proteins for destruction by the proteasome. The role of monoubiquitination is less understood, although a function in membrane trafficking is emerging, at least in yeast. Here we report that a short amino-acid stretch at the carboxy-termini of the monoubiquitinated endocytic proteins Eps15 and eps15R is indispensable for their monoubiquitination. A similar sequence, also required for this modification, is found in other cytosolic endocytic proteins, such as epsins and Hrs. These sequences comprise a protein motif, UIM (ref. 6), which has been proposed to bind to ubiquitin. We confirm this for the UIMs of eps15, eps15R, epsins and Hrs. Thus, the same motif in several endocytic proteins is responsible for ubiquitin recognition and monoubiquitination. Our results predict the existence of a UIM:ubiquitin-based intracellular network. Eps15/eps15R, epsins and Hrs may function as adaptors between ubiquitinated membrane cargo and either the clathrin coat or other endocytic scaffolds. In addition, through their own ubiquitination, they may further contribute to the amplification of this network in the endocytic pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor.

DNA methylation of tumor suppressor genes is a frequent mechanism of transcriptional silencing in cancer. The molecular mechanisms underlying the specificity of methylation are unknown. We report here that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters and that hypermethylation contributes to its leukemogenic potential. Retinoic acid treatment induces promoter demethylation, gene reexpression, and reversion of the transformed phenotype. These results establish a mechanistic link between genetic and epigenetic changes during transformation and suggest that hypermethylation contributes to the early steps of carcinogenesis.

Azacitidine↗

PubMed2002

Mechanisms through which Sos-1 coordinates the activation of Ras and Rac.

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Kinetic heterogeneity of an experimental tumour revealed by BrdUrd incorporation and mathematical modelling.

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA-BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell-cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA-BrdUrd fluorescence distribution. Sequential DNA-BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (Tpot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.

Animals↗