1,720,991 research outputs found
Viral density and virus to bacterium ratio in deep-sea sediments of the eastern Mediterranean
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A ligand-inducible anaplastic lymphoma kinase chimera is endocytosis impaired
No full textLigand-induced membrane trafficking of the anaplastic lymphoma kinase (ALK) was studied using a chimeric receptor in which the extracellular and transmembrane domain of ALK was substituted for the corresponding regions of epidermal growth factor receptor (EGFR). Wild-type EGFR, EGFR/ALK and an EGFR/ALK kinase negative mutant were independently expressed in mouse NR6 fibroblasts. The capacity of EGFR/ALK to mediate [125I]-EGF internalization, receptor degradation and downregulation, which has never been previously described, was assayed. The rate of [125I]-EGF-induced internalization mediated by the cytoplasmic domain of ALK was reduced several fold compared with the wild-type EGFR. The low rate of EGF internalization promoted by EGFR/ALK correlated with an impaired degradation and downregulation of the receptor and indicate that ALK is not subject to traditional mechanisms used to regulate receptor tyrosine kinase function. Accordingly, ALK-activated intracellular domain does not associate in vivo with c-cbl and does not undergo ligand-mediated ubiquitination. The current study provides new insight into the function and regulation of ALK suggesting that the relative long membrane residence of activated ALK might confers a more potent and prolonged signaling activity. Indeed NR6-EGFR/ALK cells exhibited a ∼3-fold increase in a maximal mitogenic response than NR6-EGFR
Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange
No full textFluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed. RI Bizzarri, Ranieri/H-1779-201
Probing Nuclear Localization Signal-Importin alpha Binding Equilibria in Living Cells
No full textThe regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Imp alpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Imp alpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Imp alpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Imp alpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Imp alpha and yield the first estimate of its autoinhibition energy in living cells
Tuning the transport properties of HIV-1 tat arginine-rich motif in living cells
No full textA large body of work is currently devoted to the rational design of new molecular carriers for the controlled delivery of cargoes (e.g. proteins or nucleic acids) to relevant subcellular domains, particularly the nucleus. In this article, we show that rational mutagenesis of the human immunodeficiency virus type 1 Tat-derived peptide (YGRKKRRQRRR) affords variants with finely tuned intercompartmental dynamics and controllable transport mechanisms. Our findings are made possible by the demonstration that the Tat peptide possesses two competing functionalities capable of active nuclear targeting and additional binding to intracellular moieties. By altering the competition between these two functions, we show how to control cargo localization of Tat peptide chimeras. Our investigation provides a unified, coherent description of previous conflicting in vivo and in vitro results and lets the true nature of the Tat peptide emerge
Green fluorescent protein based pH indicators for in vivo use: a review
No full textGreen fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community
Dendrimer Internalization and Intracellular Trafficking in Living Cells
No full textThe ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Recent studies demonstrate that dendrimers are capable to enter cells by endocytosis, but the intracellular pathway following their internalization remains controversial. In this study we use confocal fluorescence microscopy to elucidate the intracellular trafficking properties of PAMAM dendrimers with high spatial and temporal resolution in living HeLa cells. Macromolecules of different chemical functionality (neutral, cationic and lipidated), size (from G2 up to G6) and surface charge are investigated and their internalization properties correlated with the molecular structure. Toxicity and internalization data are discussed that allow the identification of dendrimers maximizing intracellular uptake with the minimum effect on cell viability. Time-lapse imaging and colocalization assays with fluorescent biomarkers for endocytic vesicles demonstrate that dendrimers are internalized by both clathrin-dependent endocytosis and macropinocytosis and are eventually delivered to the lysosomal compartment. Moreover we analyzed the uptake of dendrimers in additional cell lines of practical interest for therapeutic purposes. These measurements together with a direct comparison with TAT peptides demonstrate that PAMAM dendrimers possess similar properties to these widely used cell-penetrating peptides and thanks to their chemical tunability may represent a valid alternative for drug and gene deliver
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