39th Meeting of the Danish Society for Flow Cytometry
Joint meeting of the Danish Society for Cyto- and Histochemistry and DSFCM.
Advanced fluorescence imaging techniques
Location Auditorium 1-01 (Festauditoriet), Faculty
of Life Sciences.
All are welcome.
Program Chair: Lars-Inge Larsson.
16.00–16.05 Introduction by
van Deurs, Department of Cellular and Molecular Medicine, the
FRAP determination of caveolar mobility in relation to EGFR endocytosis.
DNA damage-induced cell cycle checkpoints and their dynamics in living mammalian cells.
Schulz, Plant Physiology and Anatomy Laboratory, Department of Plant
Biology, Faculty of Life Sciences.
Bioimaging with ”caged probes”.
Lagerholm, MEMPHYS – Center for
Biomembrane Physics, University of Southern
Biological Applications of Quantum Dots.
18.05–18.30 Discussion by the panel of speakers.
FRAP determination of caveolar mobility in relation to EGFR endocytosis
Department of Cellular and Molecular Medicine, the
It is well-established that following Epidermal Growth Factor (EGF) binding the EGF receptor (EGFR) becomes internalized by clathrin-dependent endocytosis. However, recently it was reported that caveolae-dependent endocytosis is involved in the uptake of EGFR at high concentrations of ligand. We have previously shown that plasma membrane caveolae are stable membrane domains not involved in constitutive endocytosis . We therefore investigated whether stimulation with high concentrations (100 ng/ml) of EGF induced mobilization of plasma membrane caveolae, either as a bulk movement of cell surface caveolae towards the interior of the cell, or as an increased turnover of caveolae at the plasma membrane . Live-cell microscopy of cells expressing GFP-Caveolin-1 as a marker for caveolae revealed that no net movement of caveolae takes place in cells stimulated with high concentrations of EGF. In addition, Fluorescence Recovery after Photobleaching (FRAP) analysis of GFP-labeled plasma membrane caveolae showed that EGF stimulation does not increase the turnover of caveolae at the plasma membrane. Both in control cells and in EGF stimulated cells, the mobile fraction of caveolae was as low as 20-30%. In contrast, we found that endocytosis of EGFR was efficiently inhibited by knockdown of clathrin heavy chain, both at high and low concentrations of EGF . Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree, and high concentrations of EGF do not mobilize caveolae.
 Thomsen, P., K.Roepstorff, M.Stahlhut, and B.van Deurs. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238-250 (2002).
 Kazazic, M., K.Roepstorff, L.E.Johannessen, N.M.Pedersen, B.van Deurs, E.Stang, and I.H.Madshus. EGF-induced activation of the EGF receptor does not trigger mobilization of caveolae. Traffic. 7, 1518-1527 (2006).
DNA damage-induced cell cycle checkpoints and their dynamics in living mammalian cells
To protect the genome against adverse effects of DNA damage, eukaryotic cells evolved surveillance pathways, so-called ‘checkpoints’ that delay cell cycle progression until the productive repair of the primary DNA lesions. Our laboratory is interested in how the key checkpoint-associated molecular network operates in its physiological environment, the nucleus of a living mammalian cell. I will discuss the current advances in real-time imaging of molecular trafficking inside the nucleus and provide evidence that interaction of distinct checkpoint complexes with the sites of DNA damage is tightly regulated in space and time. I will show that the mode of protein redistribution in and out of the damaged nuclear compartments might lead to discoveries of new functions of these factors, and thus provide a more complete picture of the basic principles of functional connection between the spatially restricted sites of DNA lesions and the pan-nuclear cell cycle effectors.
1) Lukas, C., Falck, J., Bartkova, J., Bartek, J., and Lukas, J. Distinct spatio-temporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5, 255-260 (2003).
2) Lukas, C., Melander, F., Stucki, M., Falck, J., Bekker-Jensen, S., Goldberg, M., Lerenthal, Y., Jackson, S. P., Bartek, J., and Lukas, J. Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 23, 2674-2683 (2004).
3) Lukas, J., Lukas, C., and Bartek, J. Mammalian cell cycle checkpoints: Signalling pathways and their organization in space and time. DNA Repair, 3, 997-1007 (2004).
4) Bekker-Jensen, S., Lukas, C., Melander, F., Bartek, J., and Lukas, J. Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J Cell Biol. (2), 201-211 (2005).
5) Bekker-Jensen S., Lukas, C., Kitagawa R., Kastan, M.B., Bartek, J., and Lukas, J. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol.(2), 195-206 (2006).
Bioimaging with ”caged probes”
Alexander Schulz, Plant Physiology and Anatomy Laboratory, Department of Plant Biology, KU (firstname.lastname@example.org).
Some animal cell types can form direct cytosolic connections by “tunnelling nanotubes”. They are consisting of plasma membrane-limited tubes allowing passage of cytosolic compounds and even organelles . This pathway contrast strongly to gap junctions where the connecting pore is formed by protein complexes and only solutes below 800 Da size can pass.
Plant cells are long known to be directly linked by plasma-membrane limited communication channels through the cell walls called plasmodesmata (PD). Typically, many hundred PD connect neighbouring cells and are used for the exchange of disaccharides, amino acids and ions. This transport is physiologically regulated so that individual cells can rapidly respond to environmental and endogenous changes . Regulation is by constricting the channel and involves cytoskeletal proteins at the orifices of the PD and an obligatory ER-element in PD, called desmotubule, linking the ER-system of neighbouring cells.
We have followed the regulation of transport with different biomaging techniques such as FRAP of lipophilc membrane dyes  and uncaging of fluorescent tracers . This method is certainly of interest also for the study of transport through tunnelling nanotubes. After loading of the non-fluorescent caged compound into all cells, a cell of interest can be illuminated with UV light, which uncages the compound so that it gets strongly fluorescent. Its spreading to the neighbouring cells can be quantified. Significantly, the technique is totally non-invasive. Microinjection and other invasive techniques lead to the immediate closure of plasmodesmata.
 Rustom, A., Saffrich, R., Markovic,
 Schulz A (2005) Role of plasmodesmata in solute loading and unloading. In: Plasmodesmata K Oparka, ed, Blackwell Publishing, (328p), pp 135-161.
 Martens HJ, Roberts AG, Oparka KJ, Schulz A (2006) Quantification of plasmodesmatal ER coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching (FRAP).Plant Physiology, 142: 471-480.
 Martens HJ, Hansen M, Schulz A (2004) Caged probes - a novel tool in studying symplasmic transport in plant tissues. Protoplasma 223: 63-66.
Biological applications of quantum dots
Qdots are small inorganic fluorescent nanoparticles that are very photostable, are brighter than conventional dye and protein fluorophores, are excitable over a broad wavelength range stretching from the ultraviolet up to slightly less than their emission peak, and have narrow, size-tunable emission bands (Michalet et al. 2005). The unprecedented optical properties of Qdots have led to an intense interest for their use in a range of biological applications. I will discuss general properties of Qdots and give examples of their use including for labeling mammalian cells (Lagerholm et al., 2004), for use in non-invasive animal imaging (Ballou et al., 2004) and for use in single molecule fluorescence imaging (Lagerholm et al., 2006).
1) Ballou, B., Lagerholm, B. C., Ernst, L. A., Bruchez, M. P., and A. S. Waggoner. (2004) Noninvasive imaging of quantum dots in mice. Bioconjugate Chem., 151: 79-86.
2) Lagerholm, B. C., Wang, M., Ernst, L. A., Ly, D. H., Liu, H., Bruchez, M. P., and A. S. Waggoner. (2004) Multicolor coding of cells with cationic peptide coated quantum dots. Nano Letters 10: 2019-22.
3) Lagerholm, B. C., Averett, L., Weinreb, G. E., Jacobson, K., and N. L. Thompson. (2006) Analysis method for measuring submicroscopic distances with blinking quantum dots. Biophys. J. 91, 3050-60.
4) Michalet, X., F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss. 2005. Quantum dots for live cells, in vivo imaging, and diagnostics. Science. 307:538–544.
Rev. 2 March 2007 /JKL