View source for Fast Microtubule Dynamics in Meiotic Spindles Measured by Single Molecule Imaging: Evidence that the Spindle Environment does not Stabilize Microtubules

Original Entry: Peter Foster, AP 225, Fall 2011

[[Image:Dn90fig1.png|thumb|300px| Figure 1, taken from [1].]]
[[Image:Dn90fig2.png|thumb|300px| Figure 2, taken from [1].]]
[[Image:Dn90fig3.png|thumb|300px| Figure 3, taken from [1].]]
[[Image:Dn90fig4.png|thumb|300px| Figure 4, taken from [1].]]
==General Information==
'''Authors''': Daniel J. Needleman, Aaron Groen, Ryoma Ohi, Tom Maresca, Leonid Mirny, and Tim Mitchison

'''Publication''': Needleman et al. Fast microtubule dynamics in meiotic spindles measured by single molecule imaging: Evidence that the spindle environment does not stabilize microtubules. Molecular Biology of the Cell (2010) vol. 21 (2) pp. 323

'''Keywords''': spindle, single molecule imaging, ''Xenopus laevis'', cell division
==Summary==
The meiotic spindle is a piece of cellular machinery composed of microtubules (which are in turn composed of tubulin subunits) that serves to separate chromosomes during cell division. The spindle is incredibly dense with tubulin, containing a concentration much higher than found in the cytoplasm (~60 uM vs <1uM). Different theories have been proposed as to what leads to this high tubulin concentration gradient. One such theory focuses on the spindle somehow being a stabilizing environment that prevents nonkinetochore microtubules that had been nucleated elsewhere from depolymerizing. The results of this paper cast a serious doubt on this theory. 


The experiments were carried out in ''Xenopus laevis'' egg extracts where a small amount of fluorescently labeled tubulin was added. This tubulin incorporates into the microtubules as shown in Fig 1(C). The location of each labeled tubulin could be found accurately from these images by fitting a 2D gaussian function to the intensity from each point, as shown in Fig 1(A). Individual tubulin are added and removed from the microtubules in the spindle. By tracking each of these "dots" in the image from the time it appears to the time that it disappears, one can define the lifetime of each tubulin in the spindle. A plot of the relative frequency of each lifetime vs. the lifetime is found in Fig (2). The red fit line is the equation...


<math>~~~P(v) ~ \alpha   ~ t^{-3/2}e^{-t/ \tau}</math>


where tau is 4 times the expected lifetime of a microtubule of average length. This is the equation for a biased random walk. Here, tau is an adjustable parameter and the fit in Fig 2 has tau = 72 +-3 s. Even though this is a spectacular fit to the data, the authors stress that the use of a biased random walk in this context is simply a phenomenological model that serves as an approximation to  the "true" microscopic model of tubulin dynamics.


To check and see if there was a variation in local stabilization in different parts of the spindle each image was divided into 5 regions (2 pole regions, 2 midbody regions, and 1 center region) as shown in Fig 3 (A). The ration of deaths (tubulin leaving the spindle) to births (tubulin entering the spindle) was calculated and is shown for each region in figure 3 (B). It is evident that the ratio is similar in all parts of the spindle. Fig 3 (C) shows a similar plot to the one in Fig 2, but using the data collected from each region of the spindle. The overlap of points leads further support to the notion that no region of the spindle seems to be more stable than another.


To consider the stability inside vs. outside the spindle a slightly different experimental assay was used. Instead of forming spindles in the egg extract, pellicles from ''Tetrahymena'' were added to the extracts. These pellicles are known to nucleate microtubules. Fluorescent tubulin was added, and a similar analysis was done as before. Fig 4 (B) shows the relative frequency vs. life time plot for both the spindle (green) and the pellicles (blue). The similarity between the two lends credence to the idea that the spindle is not a region of increased stability for nonkinetochore microtubules.



==Discussion==
In my mind, one of the neat things about this paper is that the data could be fit so well using a biased random walk. The spindle is an enormously complicated environment containing an incredible number of proteins that all interact in different ways. Yet, this relatively simple model does an excellent job of fitting the lifetime distribution. This should be a major clue to whomever works on developing a microscopic model  of this behavior. 


The other thing I really like about this paper is the simplicity of the method used. Nothing about it is particularly complex, needing no particularly complex equipment beyond what is commonly found in many biology labs. Yet, they use this method to cast serious doubt on a model that was used for over 15 years. Many different labs could have carried out this experiment; they just had to be clever enough to figure out how to look.

==References==
[1] Needleman et al. Fast microtubule dynamics in meiotic spindles measured by single molecule imaging: Evidence that the spindle environment does not stabilize microtubules. Molecular Biology of the Cell (2010) vol. 21 (2) pp. 323