cell news 2/2013
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Figure 2 Continuous sampling
model for ISWI chromatin
remodelers.
ISWI chromatin remodelers bind
nucleosomes transiently to iden-
tify appropriate substrates. Un-
der “housekeeping” conditions,
nucleosomes are translocated
rather rarely. Only in special
cases such as DNA replication
or repair, ISWI remodelers bind
tightly to chromatin and become
active.
nikon young scientist award of the dgz
accumulate at laser-induced DNA damage sites within seve-
ral seconds to a few minutes (Erdel and Rippe, 2011), which
would not be possible with lower concentrations. Thus, the
cell seems to fne-tune its remodeling machinery to ensure
suffcient chromatin plasticity.
References
Blosser, T.R., Yang, J.G., Stone, M.D., Narlikar, G.J., and Zhuang, X. (2009). Dynamics of
nucleosome remodelling by individual ACF complexes. Nature 462, 1022-1027.
Erdel, F., Krug, J., Längst, G., and Rippe, K. (2011a). Targeting chromatin remodelers: sig-
nals and search mechanisms. Biochim Biophys Acta 1809, 497-508.
Erdel, F., Muller-Ott, K., Baum, M., Wachsmuth, M., and Rippe, K. (2011b). Dissecting
chromatin interactions in living cells from protein mobility maps. Chromosome Ressearch
19, 99-115.
Erdel, F., and Rippe, K. (2011). Binding kinetics of human ISWI chromatin-remodelers to
DNA repair sites elucidate their target location mechanism. Nucleus 2, 105-112.
Erdel, F., and Rippe, K. (2012). Quantifying transient binding of ISWI chromatin remode-
lers in living cells by pixel-wise photobleaching profle evolution analysis. Proc Natl Acad
Sci U S A 109, E3221-3230.
Erdel, F., Schubert, T., Marth, C., Langst, G., and Rippe, K. (2010). Human ISWI chromatin-
remodeling complexes sample nucleosomes via transient binding reactions and become
immobilized at active sites. Proc Natl Acad Sci USA 107, 19873-19878.
He, X., Fan, H.Y., Narlikar, G.J., and Kingston, R.E. (2006). Human ACF1 alters the remode-
ling strategy of SNF2h. J Biol Chem 281, 28636-28647.
of the protein and the kinetic rate constants for binding to
immobile obstacles can be obtained. In FCS, the focus of a
confocal microscope is placed at a fxed position, and fuo-
rescent molecules that move in or out of the observation vo-
lume are detected (Fig. 1B). From the recorded intensity fuc-
tuations the diffusion coeffcient of the particles is obtained.
Both groups of methods provide complementary information:
FRAP is typically suited for processes that occur within se-
conds to minutes, whereas FCS has high time resolution down
to microseconds but does not work for slower processes due to
limited photostability of the available fuorophores. In the ran-
ge between hundreds of milliseconds to a few seconds, both
FRAP and FCS have diffculties to reliably resolve molecular
mobility and interactions. Since the catalytic rates measured
for chromatin remodelers
in vitro
suggest that remodeling can
occur on this timescale (Blosser et al., 2009; He et al., 2006),
I devised a method to assess processes on these scales, which
we termed Pixel-wise Photobleaching Profle Evolution Analy-
sis (3PEA). It relies on ftting the intensity distribution around
the bleach region (Fig. 1C). Using FRAP, FCS and 3PEA I stu-
died the mobility of the human Imitation Switch (ISWI)-type
chromatin remodelers Snf2H and Snf2L in living cells (Erdel
and Rippe, 2012; Erdel et al., 2010). Interestingly, ISWI remo-
delers are very mobile, bind only transiently to chromatin and
seem to translocate nucleosomes rather rarely. Only in special
cases, e.g. at replication foci or at DNA damage sites, many
remodelers bind long enough to translocate nucleosomes (Fig.
2). Given the moderate activity of remodelers within their
cellular environment it is surprising that they are expressed
at relatively high levels in the micromolar range as measured
by FCS and quantitative western blotting. This apparent con-
tradiction might be resolved when the frequency at which a
nucleosome is visited by a remodeler is calculated based on
the values obtained from fuctuation microscopy. To quickly
react upon external stimuli such as occurrence of DNA dama-
ge, a high frequency is required, which can be accomplished by
high remodeler abundance and mobility. Indeed, ISWI proteins
Housekeeping: continous sampling
Replication/repair: immobilization
Fabian Erdel studied Physics and Mole-
cular Biology at Ruprecht-Karls-Univer-
sity in Heidelberg, Germany, and at Tor
Vergata University in Rome, Italy. For his
PhD he joined the lab of Karsten Rippe
at the German Cancer Research Center
(DKFZ) and BioQuant Center in Heidel-
berg, where he applied fuorescence
microscopy based methods to study in-
teractions of chromatin remodelers in
living cells. Currently, he is working as a
postdoc in Karsten Rippe's lab with a focus on light-induced mani-
pulation of activities in the cell nucleus.
