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Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity.

ABSTRACT: New information has been obtained recently regarding microtubule organization in Xenopus extract spindles. These spindles assemble in vitro by chromatin-mediated microtubule nucleation and consist of randomly interspersed long and short microtubules with minus ends distributed throughout the spindle. Fluorescence speckle microscopy has led to the proposal that the Xenopus steady-state spindles contain two overlapping arrays of parallel or antiparallel microtubules with differing poleward-flux velocities. Although some of these features have also been reported for C. elegans female meiotic spindles, it is not clear whether they are representative of microtubule organization and dynamics in oocyte meiotic spindles. Here we examine anastral meiosis I spindles of live Drosophila oocytes expressing the microtubule plus end-tracking protein, EB1, fused to GFP, and find fluorescent particles throughout the spindle and movement toward both the poles and the equator. EB1 particle velocities, corresponding to microtubule growth rates, are similar in both directions, but slower than growth from the poles in mitotic spindles of early embryos. Meiosis I spindles yielded data from photobleaching analysis showing similar microtubule growth rates and dynamics at the poles and the equator, consistent with spindle microtubules of mixed polarity, differing from early-embryo mitotic spindles.

SUBMITTER: Liang ZY

PROVIDER: S-EPMC2701147 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity.

Liang Zhang-Yi ZY Hallen Mark Andrew MA Endow Sharyn Anne SA

Current biology : CB 20090101 2

New information has been obtained recently regarding microtubule organization in Xenopus extract spindles. These spindles assemble in vitro by chromatin-mediated microtubule nucleation and consist of randomly interspersed long and short microtubules with minus ends distributed throughout the spindle. Fluorescence speckle microscopy has led to the proposal that the Xenopus steady-state spindles contain two overlapping arrays of parallel or antiparallel microtubules with differing poleward-flux ve ...[more]

PMID: 19167226

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Project description:Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

Dataset Information

Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity.

Publications

Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity.

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