Estimating the Microtubule GTP Cap Size In Vivo

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Current biology: CB (Impact Factor: 9.57). 08/2012; 22(18):1681-7. DOI: 10.1016/j.cub.2012.06.068
Source: PubMed


Microtubules (MTs) polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin in the MT lattice. Relatively stable GTP-tubulin subunits create a "GTP cap" at the growing MT plus end that suppresses catastrophe. To understand MT assembly regulation, we need to understand GTP hydrolysis reaction kinetics and the GTP cap size. In vitro, the GTP cap has been estimated to be as small as one layer [1-3] (13 subunits) or as large as 100-200 subunits [4]. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in epithelial cells, we find on average (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ∼750 tubulin subunits. Thus, in vivo, the GTP cap is far larger than previous estimates in vitro, and ∼60-fold larger than a single layer cap. We also find that the tail of a large GTP cap promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated scaffold for tip-tracking proteins and confers persistence to assembly in the face of physical barriers such as the cell cortex.

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    • "It appears that the calponin homology domain of EB proteins bridges microtubule protofilaments and binds close to the GTP-binding site (Maurer et al. 2012). In addition, a recent study estimates that the GTP cap on microtubules in cells is actually quite large (> 700 tubulin subunits) and binds almost 300 EB1 dimers (Seetapun et al. 2012). These data suggest that growing microtubules are capable of concentrating +TIP proteins by almost 100-fold over their concentration in the cytoplasm, which bolsters the conclusion that growing microtubule plus ends act as 'magnets' or 'diffusional sinks' (Akhmanova et al. 2009) that are capable of concentrating microtubule-associated proteins in time and space. "
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    ABSTRACT: Microtubules in neurons consist of highly dynamic regions as well as stable regions, some of which persist after bouts of severing as short mobile polymers. Concentrated at the plus ends of the highly dynamic regions are proteins called +TIPs that can interact with an array of other proteins and structures relevant to the plasticity of the neuron. It is also provocative to ponder that short mobile microtubules might similarly convey information with them as they transit within the neuron. Thus beyond their known conventional functions in supporting neuronal architecture and organelle transport, microtubules may act as "information carriers" in the neuron. This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 11/2013; 129(2). DOI:10.1111/jnc.12621 · 4.28 Impact Factor
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    ABSTRACT: The 'GTP cap' of the microtubule has long been postulated to exist, but a recent experiment gives us the first quantitative measurements of the cap size in the cell.
    Current biology: CB 09/2012; 22(18):R802-3. DOI:10.1016/j.cub.2012.07.050 · 9.57 Impact Factor
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    ABSTRACT: Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells.
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