root dependence of the diffusion coefﬁcient on the molecular
mass predicts only 1.5–23 smaller diffusion coefﬁcient of
the individual protein molecules in buffer compared to GFP.
These results indicate the role of cytoplasmatic crowding on
the motion of the investigated proteins.
Although PAR-2 and CDC-37 are of similar size, PAR-2
diffusion is approximately three times slower, indicating
possible localization in a larger complex or self-association.
Similarly, the low diffusion coefﬁcient of NMY-2 may be a
consequence of association with other cytoplasmic compo-
nents and homodimerization. The possible involvement of
PAR-2 and NMY-2 in large protein complexes could be
linked to the mechanism by which they become localized to
We have shown that FCS and sFCS can be used to study the
dynamics of ﬂuorescently labeled molecules on both short
and long timescales even in such a complex and dynamic
system as a polarized embryo that will divide asymmetri-
cally, by overcoming the limitations of low statistical ac-
curacy and photobleaching. Although cortex localization of
PAR-2 depends on the presence of NMY-2 (8), our data in-
dicate that PAR-2 is not recruited to the cortex by binding to
posterior localized NMY-2 patches. By using circular sFCS,
we could show that PAR-2 dynamics are faster than NMY-2,
and further that PAR-2 distribution in the cortex is not uni-
form but heterogeneous, with a highly dynamic pattern dis-
tinct from that of NMY-2. It is therefore more likely that
NMY-2 changes the properties of the cortex in a way that
PAR-2 can associate with it, and presence of PAR-2 on the
cortex might be inhibitory for NMY-2 contractility.
Circular sFCS, with its single-molecule sensitivity and full
utilization of the ﬂuorescence signal, provides information
about the molecular dynamics and the type of motion, which
is too slow for standard FCS, and not resolvable with imag-
ing. Furthermore, sFCS provides information on spatial
correlation in addition to temporal correlation, facilitating
better characterization of transport processes in living orga-
nisms and discrimination between different models on basis
of their spatiotemporal correlation.
Measurement along a perimeter of a relatively large circle
overcomes two signiﬁcant limitations encountered in FCS
when applied to slowly moving molecules: photobleaching
accompanied by depletion of ﬂuorescent molecules in the ﬁxed
measurement area, and statistical noise due to the insufﬁcient
number of molecules crossing the measurement volume during
the measurement. In comparison to imaging, higher temporal
resolution, determined by the scanning frequency, is achieved
with sFCS. Furthermore, by using two-photon excitation one
additionally beneﬁts from the possibility of long measurement
times without disturbing the embryo development.
Future sFCS studies on other polarity proteins, along with
ﬂuorescence microscopy and RNAi experiments, will con-
tribute to a better understanding of asymmetric cell division
in C. elegans and in other systems.
To view all of the supplemental ﬁles associated with this
article, visit www.biophysj.org.
We thank Carrie Cowan and Nathan Goehring for stimulating discussions.
C.H. was supported by an Ernst Schering Foundation Postdoctoral fellow-
ship. Funding was provided by The International Human Frontier Science
Program grant No. RGP 5-2005, and Europa¨ische Fond fu¨r Regionale
Entwicklung project No. 4212-06-02.
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