Nikta Fakhri

High-resolution mapping of intracellular fluctuations using carbon nanotubes

Motors stirring within the living cell Cytoskeletal dynamics is key to cellular function. At very short time scales, thermal motions probably dominate, whereas on time scales from minutes to hours, motor-protein-12–based directed transport is dominant. But what about the times in between? Fakhri et al. tracked kinesin molecules labeled with carbon nanotubes and monitored their motion in living cells for milliseconds to hours. The kinesins motored along microtubule tracks, but sometimes moved more randomly as the tracks themselves were moved by active, larger-scale cell movements. This active “stirring” of the cytoplasm may play a role in nonspecific transport. Science, this issue p. 1031 Random active stress fluctuations, detected by tracking labeled kinesin motors, stir the cytoplasm of eukaryotic cells. 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 fluctuations.

Diameter-dependent bending dynamics of single-walled carbon nanotubes in liquids

By relating nanotechnology to soft condensed matter, understanding the mechanics and dynamics of single-walled carbon nanotubes (SWCNTs) in fluids is crucial for both fundamental and applied science. Here, we study the Brownian bending dynamics of individual chirality-assigned SWCNTs in water by fluorescence microscopy. The bending stiffness scales as the cube of the nanotube diameter and the shape relaxation times agree with the semiflexible chain model. This suggests that SWCNTs may be the archetypal semiflexible filaments, highly suited to act as nanoprobes in complex fluids or biological systems.

Brownian motion of stiff filaments in a crowded environment.

Movement in a Tight Squeeze The motion of flexible polymer chains in a dense melt or concentrated solution is described by reptation theory, in which a single chain is considered to snake back and forth inside a virtual confining tube formed by all its neighboring chains. A number of theories have been proposed for stiffer molecules, but it has been hard to obtain experimental data to determine the thermal motion of stiff filaments. Fakhri et al. (p. 1804) visualized carbon nanotubes directly as a model system for stiff polymers diffusing in a gel, and found that even a slight increase in flexibility significantly sped up diffusion of stiff filaments under confinement. The rotational diffusion constant grew linearly with the flexibility and, counterintuitively, did not depend on the degree of crowding. The thermal motion of single-walled carbon nanotubes is used to track the dynamic motion of stiff macromolecules. The thermal motion of stiff filaments in a crowded environment is highly constrained and anisotropic; it underlies the behavior of such disparate systems as polymer materials, nanocomposites, and the cell cytoskeleton. Despite decades of theoretical study, the fundamental dynamics of such systems remains a mystery. Using near-infrared video microscopy, we studied the thermal diffusion of individual single-walled carbon nanotubes (SWNTs) confined in porous agarose networks. We found that even a small bending flexibility of SWNTs strongly enhances their motion: The rotational diffusion constant is proportional to the filament-bending compliance and is independent of the network pore size. The interplay between crowding and thermal bending implies that the notion of a filament’s stiffness depends on its confinement. Moreover, the mobility of SWNTs and other inclusions can be controlled by tailoring their stiffness.

Do Inner Shells of Double-Walled Carbon Nanotubes Fluoresce?

The reported fluorescence from inner shells of double-walled carbon nanotubes (DWCNTs) is an intriguing and potentially useful property. A combination of bulk and single-molecule methods was used to study the spectroscopy, chemical quenching, mechanical rigidity, abundance, density, and TEM images of the near-IR emitters in DWCNT samples. DWCNT inner shell fluorescence is found to be weaker than SWCNT fluorescence by a factor of at least 10,000. Observable near-IR emission from DWCNT samples is attributed to SWCNT impurities.

Self-assembly of ordered nanowires in biological suspensions of single-wall carbon nanotubes

We investigate the self-assembly of ordered nanowires from length-purified single-wall carbon nanotubes (SWCNTs) in aqueous suspensions of the biological surfactant sodium deoxycholate. Macroscopically straight and nearly periodic linear arrangements of aligned individual SWCNTs are found to self-assemble in two-dimensional geometries from nanotube suspensions that are otherwise stable in the bulk, which we attribute to a dominance of surface effects under strong confinement. Directed self-assembly is explored through surface patterning, opening up new potential routes to nanotube manipulation for optical diagnostics and applications that require ordered arrangements of mutually aligned SWCNTs. The stability of these structures to thermal fluctuations and changes in solution chemistry are surveyed with near-infrared fluorescence microscopy.

Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction

Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

A surprising twist

X-ray crystallography has revealed an unusual structural element in kinesin-5 motor proteins.