Disturbance in microtubule dynamic instability

Microtubules consist of α- and β-tubulin heterodimer subunits which assemble into protofilaments. These protofilaments further form a hollow cylinder, the microtubule. Microtubules are polar structures due to the head-to-tail assembly of the tubulin heterodimers. The faster growing end of microtubules is called the plus end whereas the slower growing end is the minus end. The plus end is protected from rapid polymerisation by a GTP cap which is a ring of GTP-tubulin . However, when a new tubulin dimer is added to the plus end, GTP gets hydrolysed in the catalytic domain of α-tubulin and the tubulin subunit gets non-exchangeable. [1] Microtubules continuously undergo de- and repolymerization which is summarized as “microtubule dynamic instability”. [2] Microtubules are also stabilized by different proteins like microtubule-associated proteins (MAPs). Microtubule-related proteins often have preferential affinity for specifically modified microtubule regions, which is known as the microtubule code, including post-translational modifications like acetylation, detyrosination or polyamination. [1, 3] Furthermore, microtubules in axons exhibit specific orientation with the plus end pointing away from the soma whereas dendrites show mixed orientation of microtubules. [3] Due to the morphology of peripheral neurons possessing processes that can reach a length of more than one meter, an intact microtubule network is indispensable to ensure the supply of even the most distant parts of the neurites. [4]

- Nocodazole approach: Nocodazole is a microtubule-depolymerizing agent. Treatment with nocodazole over different time periods and subsequent quantification of microtubule mass by electron microscopy can give an insight into microtubule stability and the ratio of stabile and labile microtubule fractions. Labile microtubules will be degraded faster while stabile microtubules require more time for depolymerization. [5]

- Immunofluorescence staining for acetylated tubulin, which is a marker for stable microtubules and for tyrosinated tubulin which is a marker of labile microtubules gives insight into the composition of microtubules in cells. [3, 5]

- Fluorescence recovery after photobleaching: Cultured neurons are transfected to express fluorescently-labelled tubulin or injected with x-rhodamine-tubulin (tubulin with fluorescent-label). Multiple sites along a neurite are photobleached and fluorescence recovery after photobleaching was assessed at these sites as a measure for microtubule turnover. [3, 6]

1. Conde, C. and A. Caceres, Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci, 2009. 10(5): p. 319-332.

2. Mitchison, T. and M. Kirschner, Dynamic instability of microtubule growth. Nature, 1984. 312(5991): p. 237-242.

3. Baas, P.W., et al., Stability properties of neuronal microtubules. Cytoskeleton (Hoboken), 2016. 73(9): p. 442-60.

4. Griffin, J.W. and D.F. Watson, Axonal transport in neurological disease. Annals of Neurology, 1988. 23(1): p. 3-13.

5. Baas, P.W. and M.M. Black, Individual microtubules in the axon consist of domains that differ in both composition and stability. J Cell Biol, 1990. 111(2): p. 495-509.

6. Edson, K.J., et al., FRAP analysis of the stability of the microtubule population along the neurites of chick sensory neurons. Cell Motil Cytoskeleton, 1993. 25(1): p. 59-72.