XB-ART-49689
J Vis Exp
September 7, 2014;
(91):
e52138.
Using plusTipTracker software to measure microtubule dynamics in Xenopus laevis growth cones.
Abstract
Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics(1,2). One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein,
EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization(1). Many studies of
EB1 behavior within growth cones have used time-consuming and biased computer-assisted,
hand-tracking methods to analyze individual MTs(1-3). Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker(4), following the acquisition of high-resolution, live images of tagged
EB1 in cultured embryonic growth cones(5). This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types(6-8).
PubMed ID:
25225829
PMC ID:
PMC4189079
Article link:
J Vis Exp
Grant support:
[+]
R00 MH095768 NIMH NIH HHS
Species referenced:
Xenopus laevis
Genes referenced:
mapre1
References [+] :
Akhmanova,
Tracking the ends: a dynamic protein network controls the fate of microtubule tips.
2008,
Pubmed
Akhmanova,
Tracking the ends: a dynamic protein network controls the fate of microtubule tips.
2008,
Pubmed
Applegate,
plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.
2011,
Pubmed
Dixit,
Microtubule plus-end tracking by CLIP-170 requires EB1.
2009,
Pubmed
Lee,
The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.
2004,
Pubmed
,
Xenbase
Li,
EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells.
2011,
Pubmed
Long,
Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.
2013,
Pubmed
,
Xenbase
Lowery,
Neural Explant Cultures from Xenopus laevis.
2013,
Pubmed
,
Xenbase
Lowery,
Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth.
2013,
Pubmed
,
Xenbase
Marx,
Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.
2013,
Pubmed
,
Xenbase
Maurer,
EB1 accelerates two conformational transitions important for microtubule maturation and dynamics.
2014,
Pubmed
Mimori-Kiyosue,
The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.
2000,
Pubmed
,
Xenbase
Mitchison,
Dynamic instability of microtubule growth.
1984,
Pubmed
Myers,
Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis.
2011,
Pubmed
Nishimura,
Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.
2012,
Pubmed
Purro,
Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli.
2008,
Pubmed
Schuyler,
Microtubule "plus-end-tracking proteins": The end is just the beginning.
2001,
Pubmed
Stepanova,
Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).
2003,
Pubmed
Zanic,
Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.
2013,
Pubmed