Pac-man motility of kinetochores unleashed by laser microsurgery

LaFountain, James R.; Cohan, Christopher S.; Oldenbourg, Rudolf

doi:10.1091/mbc.e12-04-0314

Cited by 14 publications

(10 citation statements)

References 30 publications

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“…The photogenic nature of these cell preparations has lead to an instructive series of cell‐division movies, often used for educational purposes and available in the Cell Image Library (http://www.cellimagelibrary.org/) created and maintained by the American Society for Cell Biology. Combined with laser microsurgery and fluorescent speckle microscopy, time‐lapse series of LC‐PolScope images of crane fly spermatocytes have also led to important insights into mechanisms that contribute to the alignment of chromosomes at the metaphase plate (LaFountain et al, ; LaFountain and Oldenbourg, ) and to the modulation of kinetochore tension during meta‐ and anaphase (LaFountain et al, ). Yet, even though the birefringence of the spindle has long been studied using the traditional polarizing microscope and was instrumental in establishing the dynamic nature of its microtubule filaments (see above), the origin of birefringence of other cellular structures, like those highlighted in Regions 1 and 3 of Figure , is less well understood.…”

Section: Lc‐polscopementioning

confidence: 99%

Polarized light microscopy in reproductive and developmental biology

Koike-Tani

Tani

Mehta

et al. 2013

Molecular Reproduction Devel

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SUMMARY The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. It is a powerful tool used to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article, we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on: chromosome packing in the sperm head, the first zygote division of the sea urchin, and differentiation initiated by the first asymmetric cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end with new results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12–20.

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Section: Lc‐polscopementioning

confidence: 99%

Polarized light microscopy in reproductive and developmental biology

Koike-Tani

Tani

Mehta

et al. 2013

Molecular Reproduction Devel

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“…43 Moreover, under some conditions, PacMan was induced leading to plus-end shortening. 44 Moreover, the data reported here for Drosophila male meiotic cells directly correlate the up-regulation of plus end depolymerization with the naturally occurring diminution of resistance that follows chromosome separation. The molecules that govern Drosophila spermatocyte MT dynamics for chromosome movement await identification.…”

Section: Savoian / Photobleaching Spermatocyte Microtubulesmentioning

confidence: 56%

Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

Savoian

2015

J Biomol Tech

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In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.

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“…Instead, the chromosomes seem to be pushed from behind by microtubules growing and/or sliding out from the equator. The univalent X Y sex chromosomes in meiosis I crane-fly spindles move toward one pole while retaining microtubule fiber attachments to both poles [48]. The fiber on the trailing side elongates, while the leading fiber shortens.…”

Section: Anaphase In Some Cell Types Does Not Conform To the Canonmentioning

confidence: 99%

Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

Asbury

2017

Biology

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The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed.

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Pac-man motility of kinetochores unleashed by laser microsurgery

Cited by 14 publications

References 30 publications

Polarized light microscopy in reproductive and developmental biology

Polarized light microscopy in reproductive and developmental biology

Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

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