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
Time
Location Auditorium 1-01 (Festauditoriet), Faculty
of Life Sciences.
Registration Until
All
are welcome.
Program Chair: Lars-Inge Larsson.
16.00–16.05 Introduction by
16.05–16.35 Bo
van Deurs, Department of Cellular and Molecular Medicine, the
FRAP
determination of caveolar mobility in relation to EGFR endocytosis.
16.35–17.05
DNA damage-induced cell cycle checkpoints and their
dynamics in living mammalian cells.
17.05–17.35 Alexander
Schulz, Plant Physiology and Anatomy Laboratory, Department of Plant
Biology, Faculty of Life Sciences.
Bioimaging
with ”caged probes”.
17.35–18.05 Christoffer
Lagerholm, MEMPHYS – Center for
Biomembrane Physics, University of Southern
Biological Applications of Quantum Dots.
18.05–18.30 Discussion by the panel of speakers.
18.30–19.00 Refreshments.
Abstracts
FRAP determination
of caveolar mobility in relation to EGFR endocytosis
Bo van
Deurs,
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 [1]. 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 [2]. 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 [2]. 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.
References:
[1]
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).
[2]
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.
References:
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 (als@life.ku.dk).
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 [1]. 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 [2]. 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 [3] and
uncaging of fluorescent tracers [4]. 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.
References:
[1] Rustom, A., Saffrich, R., Markovic,
[2] Schulz A (2005) Role of plasmodesmata in solute
loading and unloading. In: Plasmodesmata K Oparka, ed,
Blackwell Publishing, (328p), pp 135-161.
[3] 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.
[4] 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).
References:
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