Estimating the Microtubule GTP Cap Size In Vivo

ArticleinCurrent biology: CB 22(18):1681-7 · August 2012with19 Reads
DOI: 10.1016/j.cub.2012.06.068 · Source: PubMed
Abstract
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.
    • "While the debate about the size of the stabilising cap was ongoing, microtubule end binding proteins were discovered (Beinhauer et al., 1997; Su et al., 1995 ) and later used to facilitate microtubule end tracking in living cells (Busch and Brunner, 2004; Komarova et al., 2009; MimoriKiyosue et al., 2000; Seetapun et al., 2012). Our quantitative demonstration that the EB binding region is the protective microtubule cap means that EBs can be used to monitor the momentary stability of individual microtubules. "
    [Show abstract] [Hide abstract] ABSTRACT: The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.
    Full-text · Article · Apr 2016
    • "While the debate about the size of the stabilising cap was ongoing, microtubule end binding proteins were discovered (Beinhauer et al., 1997; Su et al., 1995 ) and later used to facilitate microtubule end tracking in living cells (Busch and Brunner, 2004; Komarova et al., 2009; MimoriKiyosue et al., 2000; Seetapun et al., 2012). Our quantitative demonstration that the EB binding region is the protective microtubule cap means that EBs can be used to monitor the momentary stability of individual microtubules. "
    [Show abstract] [Hide abstract] ABSTRACT: The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.
    Full-text · Article · Apr 2016
    • "In vitro studies have identified EB1 as a factor that decreases the maturation time of growing microtubule ends [50], connecting EB1 localization and regulation of MT dynamics. The length of EB comets reflects the size of the microtubule GTF cap [48]. The shortening of microtubule comets that we observed in β-actin depleted cells indeed usually correlates with frequent switching between very short periods of growth and shortening. "
    [Show abstract] [Hide abstract] ABSTRACT: Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.
    Full-text · Article · Nov 2015
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