Biomedical subjects

Margarida Barroso

Publications and source records attributed to Margarida Barroso.

7 recordsLinked to original sources

PubMed2006

Issues in confocal microscopy for quantitative FRET analysis.

Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.

Algorithms↗

PubMed2005

Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.

F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.

Animals↗

PubMed2004

Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules.

Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30-100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35-40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a K(D) of approximately 0.5 microM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule-membrane interactions by affecting the p22-microtubule, but not the p22-membrane, association.

Amino Acid Sequence↗

PubMed2003

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements.

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.

Animals↗

PubMed2003

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Animals↗

PubMed2003

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy.

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.

Algorithms↗

PubMed2003

One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes.

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

Algorithms↗