Mechanical Properties of Xenopus Egg Cytoplasmic Extracts
Original Entry: Peter Foster, AP 225, Fall 2011
Authors: M. T. Valentine, Z. E. Perlman, T. J. Mitchison, and D. A. Weitz
Publication:Perlman et al. Mechanical properties of Xenopus egg cytoplasmic extracts. Biophysical journal (2005) Volume 88, Issue 1, 680-689
This paper deals with the mechanical properties of cytoplasmic extracts from Xenopus laevis (African tree from). Xenopus extract is a common system because it's relatively easy to crush the eggs and extract the cytoplasm and each egg contains everything needed for ~30 divisions. Because of how well used this system is, it's interesting to consider the mechanical properties. This egg extract is used as an example system to try to study cytoplasm mechanics in general. In the paper, conventional macroscopic rheological measurements are made as well as microscopic microrheological measurements.
Figure 1 shows results for the time evolution of the elastic moduli (top panel) and the viscous moduli (lower panel) obtained using a conventional strain controlled rheometer. Each of the curves shown represents a separate measurement and it is evident that there is substantial variation. The common theme between all of the runs is that the extract forms a gel that acts like a soft solid (G"/G' ~ 0.3-0.5) The solid like behavior comes from the cytoskeleton. When the extract was treated with latrunculin (which depolymerizes the actin network), the extract doesn't develop a measurable elastic modulus. When the extract is treated with nocodazole (which disrupts the microtubule network), the extracts show a weak viscoelectic behavior but with an elastic modulus of 0.1–0.5 Pa (~10 times weaker than the untreated samples)
Figure 2 shows the results from microrhelogical measurements. A 1um colloidal bead that was passivated by PEG coating was embedded in the extract, and video microscopy was used to track the bead's movement. Figure 2 is a plot of <r^2> versus time. The different symbols represent different chemical treatments. The squares represent untreated extract, the circles represent extract treated with 30uM latrunculin (latrunculin disrupts actin filaments), the triangles pointing down represent extract treated with 10 uM nocodazole (nocodazole interferes with the formation of micrtubules), the diamonds represent extract treated with 10uM taxol (taxol promotes microtubule stability) and the arrows pointing up represent extract treated with 500 nM taxol. The open symbols represent measurements made 10 minutes after incubation, while the solid symbols represent measurements made 30 minutes after incubation. As can be seen in the figure, the addition of chemicals that disrupt the cytoskeletal network as well as agents that stabilize microtubules seem to have little effect on the bead's motion. Fits were made to the equation <r^2> ~ tau^alpha, resulting in values for alpha between 0.7 and 0.95. If alpha = 1, then the motion is pure diffusion. These slightly lower values for alpha are subdiffusive, but show that viscosity is much more important that the elastic properties of the gel on the micron length scale. Based on these measurements, the authors estimate a value of for the extract's viscosity as ~10-30 mPa-s.
Left to itself, the extract will contract into a dense pellet (Fig 3(a)). In figure 3, measurements were made of the area of this dense pellet as a function of time with added agents to selectively stabilize or destabilize different cytoskeletal elements. In figure 3 (b), we see the results for the actin network. When the network is disrupted using latrunculin, the network is found to never contract, while when phalloidin was added to stabilize the actin network, the contraction was accelerated relative to the untreated sample. In figure 3(c) we see the results for the microtubule network, When nocodazole is added to destabilize microtubules, there is a lowering in the rate of contration, with a similar overall time evolutions. Stabilizing the microtubules with taxol, leads to a temporal delay in the onset of contraction.
What I like about this paper is how it spans length scales from the microscopic to the macroscopic and shows that the behavior of the material is not the same on all length scales. I've read microrheological papers that claim one of the advantages of microrhelogical measurements is that you need a signifigantly smaller volume of your sample compared to conventional rheology. However, it's important to note (as in this paper) that it's definitely possible for the material's behavior to be quite different on these different size scales. This is also an excellent paper that shows how tools traditionally used in physics and chemistry can be used to gain knowledge about a biological system.
Perlman et al. Mechanical properties of Xenopus egg cytoplasmic extracts. Biophysical journal (2005))