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We thank the personnel of the Invasive Spartina Project,
including P. Olofson, E. Grijalva, D. Kerr, I. Hogle, W. Thornton,
M. Latta, and others, who provided data on Spartina
distributions, clapper rail populations, and costs of eradication
and restoration efforts. We thank D. Kling for research
assistance and P. Reynolds for comments on the manuscript.
We thank the California State Coastal Conservancy and the
California State Wildlife Conservation Board for use of their data.
The data used for this paper are summarized in table S1 (24)
and are given in more detail in (24). This research was supported
by NSF grant no. DEB 1009957 to A.H., E.D.G., J.N.S., A.L.,
Materials and Methods
Figs. S1 to S6
Tables S1 to S4
13 January 2014; accepted 23 April 2014
High-resolution mapping of
intracellular fluctuations using
Nikta Fakhri,1 Alok D. Wessel,1 Charlotte Willms,1 Matteo Pasquali,2
Dieter R. Klopfenstein,1 Frederick C. MacKintosh,3 Christoph F. Schmidt1*
Cells are active systems with molecular force generation that drives complex dynamics
at the supramolecular scale. We present a quantitative study of molecular motions
in cells over times from milliseconds to hours. Noninvasive tracking was accomplished
by imaging highly stable near-infrared luminescence of single-walled carbon nanotubes
targeted to kinesin-1 motor proteins in COS-7 cells. We observed a regime of active
random “stirring” that constitutes an intermediate mode of transport, different from
both thermal diffusion and directed motor activity. High-frequency motion was found
to be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics
dominated. In addition to directed transport along microtubules, we observed strong
random dynamics driven by myosins that result in enhanced nonspecific transport.
We present a quantitative model connecting molecular mechanisms to mesoscopic
The cytoplasm of eukaryotic cells is a highly dynamic composite polymer material. Its mechanical properties are dominated by protein polymers: microtubules (MTs), F- actin, and intermediate filaments (1–4).
Metabolism maintains a chemical nonequilibrium that energizes this mechanical framework of
cells. Dominant driving forces stem from the polymerization of actin and tubulin and from motor
proteins, both deriving energy from nucleotide
triphosphate hydrolysis (5, 6). Molecules self-organize into complex machineries on all scales
to drive functions as various as intracellular transport, cell locomotion, and muscle contraction.
Understanding these machineries requires ob-
serving intracellular dynamics from molecular
to macroscopic scales. Fluorescence micros-
copy allows labeling of specific targets, but it
has been impossible to achieve long-term track-
ing of single molecules because of the fluorescent
background in cells and fluorophore instabilities.
Observations of intramolecular dynamics have
often used mesoscopic endogenous particles
or ingested beads (7, 8) instead of molecular
Generally, dynamics in cells are scale-dependent.
At short times (microseconds to milliseconds),
thermal motions should dominate. Between milliseconds and seconds, thermal diffusion might
still be relevant, but there is mounting evidence,
both in vitro and in vivo, that the motion of larger
objects couples to myosin-driven stress fluctuations in the cytoskeleton (9, 10). Here, temporal
fluctuations, reminiscent of thermal diffusion in
liquids, can arise from nonequilibrium dynamics
in the viscoelastic cytoskeleton (11). On longer
time scales, from minutes to hours, directed transport and larger-scale collective motions typically
dominate. The motion of probe particles tracked
inside cells has been classified as subdiffusive,
diffusive, or superdiffusive. Such classifications,
however, obscure the distinction between thermally driven and nonequilibrium fluctuations
and are inadequate to identify intracellular material properties.
Motor proteins are good reporters of dynamics
from the molecular scale upward because they
drive many cellular motions. Kinesins and myo-
sins have been extensively studied in vitro (12, 13),
Here, we used single-walled carbon nanotubes
(SWNTs) as a tool for high-bandwidth intracellular
tracking. SWNTs are stiff quasi–one-dimensional
tubular all-carbon nanostructures with diame-
ters of ~1 nm and persistence lengths above
10 mm (17). Individual semiconducting SWNTs
luminesce with large Stokes shifts in the near-
infrared (900 to 1400 nm) (18). This window is
virtually free of autofluorescence in biological
tissues. Fluorescence emission is highly stable with
no blinking and negligible photobleaching (19, 20)
(fig. S1), allowing for long-term tracking (21). The
fluorescence lifetime is short [~100 ps (22)] so
that high excitation intensities allow millisecond
To track the dynamics of the cytoskeleton
without introducing invasive probes, we specifically targeted short SWNTs (~100 to 300 nm;
fig. S2) to the endogenous kinesin-1 motor Kif5c
in cultured COS-7 cells (see supplementary materials). Kif5c functions as a cargo transporter in
cells (23). We dispersed SWNTs by wrapping with
short DNA oligonucleotides and used HaloTag
(24) to covalently attach SWNTs specifically to
full-length kinesins expressed in the cells (Fig. 1,
A and B). We used an additional green fluorescent protein label to confirm localization and
motility of the motors on MTs (figs. S3 and S4
and movie S1). Tracking motor proteins makes it
possible to observe several types of dynamics.
Besides observing directed kinesin-driven transport on MTs, it is possible to directly observe
fluctuations of the MT network because a moving
kinesin must be bound to a MT. The MT tracks
are embedded in the viscoelastic actin cytoskeleton, which in turn fluctuates as a result of
stresses generated by cytoplasmic myosins (Fig.
1C) (25, 26).
The high photostability of SWNTs made it
possible to introduce only a small number, around
100 per cell, and still track individual SWNTs
1Drittes Physikalisches Institut–Biophysik, Georg-August-Universität, 37077 Göttingen, Germany. 2Department
of Chemical and Biomolecular Engineering, Department
of Chemistry, Smalley Institute for Nanoscale Science and
Technology, Rice University, Houston, TX 77005, USA.
3Department of Physics and Astronomy, Vrije Universiteit,
1081 HV Amsterdam, Netherlands.
*Corresponding author. E-mail: christoph.schmidt@phys.
uni-goettingen.de (C.F.S.); email@example.com (F.C.M.)