Stability properties of neuronal microtubules

Abstract

Neurons are terminally differentiated cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression-bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. The stability properties of neuronal microtubules are commonly discussed in the biomedical literature as crucial to the development and maintenance of the nervous system, and have recently gained attention as central to the etiology of neurodegenerative diseases. Drugs that affect microtubule stability are currently under investigation as potential therapies for disease and injury of the nervous system. There is often a lack of consistency, however, in how the issue of microtubule stability is discussed in the literature, and this can affect the design and interpretation of experiments as well as potential therapeutic regimens. Neuronal microtubules are considered to be more stable than microtubules in dividing cells. On average, this is true, but in addition to an abundant stable microtubule fraction in neurons, there is also an abundant labile microtubule fraction. Both are functionally important. Individual microtubules consist of domains that differ in their stability properties, and these domains can also differ markedly in their composition as well as how they interact with various microtubule-related proteins in the neuron. Myriad proteins and pathways have been discussed as potential contributors to microtubule stability in neurons. © 2016 Wiley Periodicals, Inc.

Keywords: +tip; Alzheimer's disease; CAMSAP; MAP6; acetylation; axon; dendrite; detyrosination; fidgetin; katanin; labile; microtubule; microtubule stability; neurodegeneration; neuron; nocodazole; polyamination; spastin; stable; tau; taxol; tubulin.

Figure 1. Microtubule organization in the vertebrate neuron

Schematic of typical vertebrate neuron with one axon and multiple dendrites. Microtubules are nearly uniformly oriented in the axon, and non-uniformly oriented in the dendrites. Microtubules in both the axon and the dendrites consist individually of a stable domain (shown in red) and a labile domain (shown in yellow), with the labile domain toward the plus end of the microtubule. Short mobile microtubules in the axon are entirely stable. Microtubules in the axon and dendrites vary in their length, with none of them attached to the centrosome. In the axon, a higher percentage of the total microtubule mass is stable compared to the situation in the dendrite.

Figure 2. Stability domains of axonal microtubules and related molecules and modificiations

Microtubules in the axon individually consist of a stable domain toward the minus end of the microtubule and a labile domain toward the plus end of the microtubule. Panel A schematically indicates the two domains, which compositionally differ in their levels of post-translationally acetylated and detyrosinated tubulin subunits. Modified subunits (shown with green highlighted tubulin ‘tails’) are enriched in the stable domain while unmodified subunits (shown with orange highlighted tubulin ‘tails’) are enriched in the labile domain. The tip of the labile domain (plus end of the microtubule) is associated during bouts of microtubule assembly with plus-tip proteins such as EB1, EB3, CLIP and CLASP. The tip of the stable domain (minus end of the microtubule) is associated with minus-tip proteins termed CAMSAPs, which often take on a linear structure as they compete for tubulin subunits during the formation of a cap that prevents microtubule disassembly from the minus end. The two domains differ in their interactions with various microtubule-related proteins, such as the severing proteins katanin and spastin which target stable domains and the severing protein fidgetin, which targets labile domains. Panel B shows a more detailed view of tubulin post-translational modifications (detyrosination, acetylation, phosphorylation, polyglycylation and polyglutamylation) and where they occur in the tubulin subunits comprising the microtubule lattice. Note: In dendrites, the stable and labile regions of microtubules do not differ in such a pronounced way in their content of post-translationally modified tubulins, as is the case in the axon.

Figure 3. Hypothetical mechanisms of microtubule loss from neurons during neurodegenerative diseases

Panel A shows the normal situation in the axon with individual microtubules displaying stable and labile domains. Panel B shows a scenario for potential loss of microtubule mass during neurodegeneration in which the stable domains are gradually destabilized, thus shifting individual microtubules to shorter stabile domains and longer labile domains that undergo greater depolymerization. Panel C shows enhanced depolymerization of the labile domains, without destabilization of the stable domains. Panel D shows microtubule loss due to increased microtubule severing. Severing in the labile domain results in complete depolymerization of the resulting labile microtubule fragments. Severing in the stable domain produces stable fragments with some accompanying depolymerization during the severing event. These three mechanistic possibilities could also apply to microtubule loss in dendrites.

Figure 4. Nocodazole-based approach for assaying stable and labile microtubule fractions in cultured neurons

Top three panels on the right schematically show electron microscopy experiment in which the labile domain of the axonal microtubule is preferentially labeled by colloidal gold secondary antibody and tyrosinated tubulin primary antibody. After 15–30 minutes in nocodazole, the labile domains are entirely depolymerized, leaving behind only the stable domains. After an additional few hours in drug, the stable domain is reduced but much more slowly than the labile domain was lost. The graph to the left of these panels shows a hypothetical quantification of data derived from this kind of experiment in which the microtubule levels are plotted against time in drug. A biphasic loss of microtubule mass is revealed with a rapidly lost labile fraction and a slowly depolymerizing stable fraction. The lower panel to the left shows a parallel immunofluorescence experiment in which the labile domains (rich in tyrosinated tubulin) are depolymerized by 15–30 minutes in the drug, leaving behind the stable domains (rich in detyrosinated tubulin). The lower panel to the right shows the same experiment, as assessed by Western blotting rather than microscopy.

Figure 5. Immuno-microscopy-based approaches for assaying stable and labile microtubule fractions in cultured neurons

Top panel shows a hypothetical triple-label immunofluorescence experiment in which cultured neurons were pre-extracted in a microtubule-stabilizing buffer (to release free tubulin) prior to fixation, and then labeled with antibodies to total tubulin (with green secondary antibody), tyrosinated tubulin (with red secondary antibody), and acetylated tubulin (with blue secondary antibody). Total tubulin is relatively evenly distributed in dendrites, the axon, and soma, with microtubules also extending into the growth cone at the tip of the axon. Tyrosinated tubulin is relatively enriched in the dendrites, soma and growth cone microtubules. Acetylated tubulin is relatively enriched in the axonal shaft (which would also be true of detyrosinated tubulin, not shown in figure). The lower panel shows electron microscopy of axonal microtubule labeled with primary antibody to tyrosinated tubulin and secondary antibody conjugated to colloidal gold. The labile domain is densely labeled while the stable domain is unlabeled.

Figure 6. FRAP-based approach for assaying stable and labile microtubule fractions in cultured neurons

In the FRAP-based approach, cultured neurons are transfected to express fluorescently-tagged tubulin, with sufficient time allowed for the tagged tubulin to incorporate into the stable and labile microtubule fractions. Then, a bleached zone is created in the axon, after which the return of fluorescence is recorded through digital imaging. In theory, after a very rapid initial influx of free fluorescent tubulin (not shown), the fluorescence is recovered biphasically, with the first phase returning in 15–30 minutes, and the rest returning over a matter of hours. These two phases would correspond to the labile and stable microtubule fractions, respectively. FRAP has been used in the past to study microtubules in cultured neurons, but the more sophisticated use of the method to reveal stable and labile fractions remains hypothetical, as is the predicted quantification, shown graphically at the bottom of the schematic illustrations of the recovery phases. A notable shortcoming of this approach is the mobility of fluorescent microtubules into the bleached region from the fluorescent zones flanking the bleached region, as indicated in the third illustration, which could theoretically contribute to the recovery of fluorescence of the bleached zone as much as microtubule dynamics.