Speaker: Nikta Fakhri
Department: MIT department of Physics
Location: TU Delft
Date: October 28, 2016
Author: Teun Huijben
Nikta beging her talk by starting to discuss how we think about the cytoskeleton. We all imagine the cytoskeleton as a network of fibers scattered throughout the cell. The network is dynamic which means that it changes over time, since it grows and shrinks constantly by hydrolyzing ATP. However, seen over long timescales, the cytoskeleton can assumed to be in a steady state. Since the cytoskeleton is quite rigid and stable and is entangled throughout the entire cell, from a physical point of view it can therefore be seen as a gel, referred to as the actin-myosin gel. Gels have remarkable physical properties, but are never studies in cells in vivo. Nikta Fakhri and her group invented a new technique to quantitatively study the behaviour of this gel-like cytoskeleton.
Motor proteins like kinesin and myosin are a good choice to study the behaviour of the cytoskeleton. However, to use fluorescent microscopy for this purpose brings up multiple problems: fluorophores are not stable, they bleach, have a low signal to noise ratio because of the background in cells and they can only be used on short timescales. Therefore Nikta and her colleagues propose a new probe: single-walled carbon nanotubes (SWNTs, see figure 1).
figure 1: Single-walled carbon nanotube.
SWNTs have multiple advantages over conventional fluorophores. First of all they behave as semiconductors and have a large Stokes shift. They emit a narrow band near infrared light which makes it perfect for biological samples, because this wavelength is free of autofluorescence in cells. They do not blink, are not prone to photobleaching, have a long lifetime so can be used for long-term tracking. In addition, they emit only for a very short time enabling a high sampling frequency. Moreover SWNTs have good mechanical properties, they fit through small places, are very stiff and have tunable parameters (length, diameter and persistence length).
To study the cytoskeleton they attached the SWNTs to kinesin motors (figure 2). Hereby they can follow the motors very precisely over long timescales when they move throughout the cell. Roughly 100 labeled kinesin motors were added to a cell and they could be visualized for more than 1 hour with a high temporal resolution of 5 ms between each frame.
figure 2: SWNT covalently attached to a kinesin motor. In this way the motor can be followed while walking along the microtubule.
With this technique the movement of kinesin was studied on different time-scales. In the longer times (250 ms) the kinesin moves in a directed manner, which makes sense because the motor walks along a microtubule and will therefore move along a trajectory (figure 3). On the other hand, on very short time scales (5 ms) the kinesin motors moved in a very stochastic manner around an average center position. This is because of the thermal diffusive motion of the molecules.
figure 3: SWNT-labeled Kinesin movement on different timescales. With a frame time of 250 ms the kinesin moves along the microtubule (black trajectory) after initial random movement (green). A frame time of 5 ms shows the stochastic kinesin movement on the short time scales caused by thermal motion (red).
However, these findings on the long and short timescales were already known. The behaviour of interest lies in the interval between this. On the intermediate timescales they found that the kinesin is neither moving stochastically nor in a directed manner. It was moving in some random way with a low frequency, as expected from the hypothesis that the cytoskeleton behaves as a gel. All the interaction between the microtubules, actin filaments, myosin and kinesin motors will dynamically stir the cytoskeleton around in a non-equilibrium motion. This gives rise to a new regime of motion between the random thermal diffusion and directed transport. Nikta and her group provided hereby a new technique to quantitatively study this new regime in living cells. Which I found interesting, since I never thought of gels when thinking about the cytoplasm.
 Single-walled carbon nanotube:
[2,3] N Fakhri et al. High-resolution mapping of intracellular fluctuations using carbon nanotubes. Science 344 (2014)