1,548 research outputs found
In Vivo Imaging of Single-Molecule Translocation through Nuclear Pore Complexes by Pair Correlation Functions
No full textNuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu.The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which single particle tracking (SPT) has given results that cannot be confirmed by traditional single-point FCS measurements because of the lack of spatial resolution.We show that obstacles to molecular flow can be detected and that the pCF algorithm can recognize the heterogeneity of protein intra-compartment diffusion as well as the presence of barriers to transport across NE
Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time
No full textL'epistolario Cardarelli-Bacchelli (1910-1925). L'archivio privato di un'amicizia poetica, a cura di Silvia Morgani, Perugia, Morlacchi Editore, 2014, pp. 564.
No full textL'articolo è la recensione a un'edizione di lettere, scritte da Vincenzo Cardarelli a Riccardo Bacchelli, uscita nel 2014. Nel volume si raccolgono 199 documenti che contribuiscono a indagare l'intenso e proficuo rapporto intellettuale tra i due autori e a fornire una chiave di lettura inedita su eventi ed episodi culturali del tempo
Super-Resolution by Feedback Imaging: Mechanisms of Translocation through the Nuclear Pore Complex
No full textFluorescence Correlation Spectroscopy of Intact Nuclear Pore Complexes
No full textAbstractNo methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions
Analytical problems in the definition of material balances in the incineration of urban sludge
No full textDifferent digestion methods for the determination of heavy metals in urban sludges and its ashes are investigated utilizing two reference materials. The reference sludge and a real urban sludge are then utilized for incineration experiments to evaluate environmental impact both of residual ashes and of fumes produced during incineration. Material balances are performed to evaluate the distribution of heavy metals during the incineration experiments
The role of cytoskeleton networks on lipid-mediated delivery of DNA
No full textBackground: Lipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors. Results: In this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome–DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes. Conclusion: Collectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.Fil: Coppola, Stefano. Sapienza University of Rome. Dipartimento di Medicina Molecolare; ItaliaFil: Cardarelli, Francesco. Istituto Italiano di Tecnologia. Center for Nanotechnology Innovation at NEST; ItaliaFil: Pozzi, Daniela. Sapienza University of Rome. Dipartimento di Medicina Molecolare; ItaliaFil: Estrada, Laura Cecilia. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; ArgentinaFil: Digman, Michelle. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados UnidosFil: Gratton, Enrico. University of California. Department of Biomedical Engineering. Laboratory for Fluorescence Dynamics; Estados UnidosFil: Bifone, Angelo . Istituto Italiano di Tecnologia. Center for Nanotechnology Innovation at NEST; ItaliaFil: Marianecci, Carlotta. Sapienza University of Rome. Dipartimento di Chimica e Tecnologie del Farmaco; ItaliaFil: Caracciolo, Giulio. Sapienza University of Rome. Dipartimento di Medicina Molecolare; Itali
Capturing directed molecular motion in the nuclear pore complex of live cells
No full textNuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleo-cytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinÎ21 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs
Super-Resolution in a standard microscope: from fast fluorescence imaging to molecular diffusion laws in live cells
No full textA major challenge of present (and future) biophysics is to quantitatively study how molecular subensembles dynamically interact to fulfill their physiological roles in living cells and organisms. This is the classic research target of molecular cell biology and quantitative fluorescence microscopy: individual molecular components (proteins, lipids, etc.) are cloned/purified/synthesized, tagged, and analyzed. In this context, high-speed single-particle tracking (SPT) techniques play a crucial role and yield information at the nano-meso-scale on the dynamics and interactions of several molecular cellular components. Based on SPT, for instance, Kusumi and coworkers thoroughly investigated the compartmentalization of the fluid plasma membrane into submicron domains throughout the cell membrane and the hop diffusion of many relevant molecules (for a review see Kusumi et al. 2005a, 2005b; 2010). Widespread exploitation of the SPT approach is hindered, however, by at least four characteristics that, at present, appear unavoidable: (1) the experiment relies on production, purification, and labeling of a single molecule with a suitable marker particle (e.g., a gold colloidal particle or quantum dot); (2) the usable labels are rather large on the molecular scale and can induce cross-linking of target molecules or steric hindrance effects, thus affecting the biological function under study; (3) the size (colloidal gold particles typically used have diameters of 20-40 nm)
and chemical nature of the label typically prevents the application of SPT measurements to intracellular molecules; (4) a large number of single-molecule trajectories must be recorded to fit statistical criteria, thus dramatically increasing the time required to gather and analyze SPT data. In regard to these SPT drawbacks, fluorescence correlation spectroscopy (FCS) is a very attractive alternative approach. In fact, thanks to its intrinsic single-molecule sensitivity even in the presence of many molecules, it can easily afford the required statistics in a reasonable amount of time. FCS can be performed well with genetically encoded FPs and, in general, with fluorophores not particularly bright. The basic principle of fluctuation analysis is to decrease the detection volume so that small numbers of fluorescent molecules are excited at the same time. Molecules stochastically cross the open detection volume defined by the laser beam, leading to a fluctuating occupancy that follows Poisson statistics (which in turn obeys the relation that the variance is proportional to the average number of observed molecules). The underlying molecular dynamics are extracted as characteristic decay times through fluctuation correlation analysis. In its classic view, FCS is used as a “local” measurement of the concentration and residency time of molecules present in the diffraction-limited focal area defined by the laser beam. Many efforts targeted the extension of the FCS principle to the spatial scale. For instance, the focal area was duplicated (Ries and Schwille 2006), moved in space in laser “scanning” microscopes (Berland et al. 1996; Ruan et al. 2004; Heinemann et al. 2012; Cardarelli et al. 2012a, 2012b; Di Rienzo et al. 2014b), or combined with fast cameras (Kannan et al. 2006; Unruh and Gratton 2008; Di Rienzo et al. 2013b, 2014a). Using these “spatiotemporal” correlation approaches, diffusion constant, concentration, and partitioning coefficient of several membrane components were measured on both model membranes and actual biological ones (Schwille et al. 1999; Weiss et al. 2003). An alternative way to sample both time and space is based on the size change of the focal area. For instance, it was demonstrated that molecular FCS-based diffusion laws can be recovered by performing fluctuation analysis at various spatial scales larger than the laser focal area (Wawrezinieck et al. 2005; Lenne et al. 2006). Based on these data, inferences were drawn about the dynamical organization of cell membrane components by extrapolation below the diffraction limit. Along this reasoning, similar studies can be performed by downsizing the focal area: recent reports successfully exploited the ~30 nm focal area attained by stimulated emission depletion (STED) (Hell and Wichmann 1994) to directly probe the nanoscale dynamics of membrane components in a living cell by fluctuation analysis (Eggeling et al. 2009; Mueller et al. 2011; Sezgin et al. 2012). In all the FCS experiments described, however, the size of the focal spot represents a limit in spatial resolution that cannot be overcome. In addition, the information collected always requires assumptions and models that describe the molecular dynamics under study. These models must then be translated into equations to be fitted to FCS measurements. In an effort to overcome these limitations an approach based on spatiotemporal image correlation spectroscopy (Hebert et al. 2005) method will be discussed here, that is suitable for the study of the dynamics of fluorescently tagged molecules in live cells with high temporal (up to 10−4 s for images and 10−6 for line scans) and spatial (well below the diffraction limit) resolution (schematic representation in Figure 2.1)
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