Micromanipulation of Chromosomes in Insect Spermatocytes

Lin, Nicolas K.H.; Nance, Ryder; Szybist, Jane; Cheville, Alan; Paliulis, Leocadia V.

doi:10.3791/57359

Cited by 9 publications

(6 citation statements)

References 26 publications

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“…S3 ). This is surprising as force-induced detachments from kinetochores occur in vitro ( Akiyoshi et al, 2010 ) and in meiotic insect cells ( Nicklas, 1967 ; Nicklas and Koch, 1969 ; Paliulis and Nicklas, 2004 ; Lin et al, 2018 ). This difference could, for example, arise from variations in force application, or in the physical properties or architectures of their kinetochores ( Cheerambathur et al, 2017 ; Auckland et al, 2017 ; Agarwal et al, 2018 ; Yoo et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we use glass microneedles to directly exert force on individual k-fibers inside mammalian cells and determine how their structure and dynamics remodel under sustained force. Inspired by experiments in insect spermatocytes ( Nicklas and Staehly, 1967 ; Nicklas, 1997 ; Lin et al, 2018 ), we sought to adapt microneedle manipulation to pull on k-fibers in mitotic mammalian cells for many minutes while monitoring their dynamics with fluorescence imaging. We show that forces applied for minutes regulate k-fiber dynamics at both ends, causing k-fiber lengthening, but do not cause sliding of the microtubules within them.…”

Section: Introductionmentioning

confidence: 99%

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Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Long

Suresh

Dumont

2020

Journal of Cell Biology

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At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Long

Suresh

Dumont

2020

Journal of Cell Biology

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“…Living cell preparations of adult male testes were prepared at room temperature according to the method of Lin et al [10]. Primary spermatocytes undergoing meiosis were filmed across multiple focal planes using a Zeiss Opton inverted phase contrast microscope as described in Lin et al [10], or using a Zeiss Observer microscope equipped with a 40X, 0.65 N.A. objective and an Axiocam 503 monochrome camera.…”

Section: Living Cell Preparationsmentioning

confidence: 99%

Chromosome number, sex determination, and meiotic chromosome behavior in the praying mantid Hierodula membranacea

et al. 2022

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Praying mantids are important models for studying a wide range of chromosome behaviors, yet few species of mantids have been characterized chromosomally. Here we show that the praying mantid Hierodula membranacea has a chromosome number of 2n = 27, and X1X1X2X2 (female): X1X2Y (male) sex determination. In male meiosis I, the X1, X2, and Y chromosomes of H. membranacea form a sex trivalent, with the Y chromosome associating with one spindle pole and the X1 and X2 chromosomes facing the opposite spindle pole. While it is possible that such a sex trivalent could experience different spindle forces on each side of the trivalent, in H. membranacea the sex trivalent aligns at the spindle equator with all of the autosomes, and then the sex chromosomes separate in anaphase I simultaneously with the autosomes. With this observation, H. membranacea can be used as a model system to study the balance of forces acting on a trivalent during meiosis I and analyze the functional importance of chromosome alignment in metaphase as a preparatory step for subsequent correct chromosome segregation.

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Section: Living Cell Preparationsmentioning

confidence: 99%

“…Testes were removed from the abdomens of E. latipes males and transferred to a culture chamber (Lin et al 2018) under a layer of Kel-F Oil #10 (Ohio Valley Specialty Company, Marietta, Ohio). Testes contents were spread thinly on a coverslip under oil, as described in Lin et al (2018). Living meiosis I spermatocytes were imaged using a Zeiss inverted microscope equipped with a 100X 1.25 NA phase-contrast, oil-immersion objective and an Infinity 1 camera with Infinity Analyze software or a Nikon Eclipse TS100 microscope equipped with a 100X, 1.25 NA phase-contrast, oil-immersion objective and a Spot RT monochrome camera (Diagnostic Instruments Inc.) with Spot Basic 3.5.7 software.…”

Section: Living Cell Preparationsmentioning

confidence: 99%

Segregation of the univalent X chromosome in the wide-footed treehopper Enchenopa latipes (Say 1824)

et al. 2022

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In metaphase I, autosomal bivalents align on the metaphase plate, while naturally-occurring univalent sex chromosomes can display a number of different behaviours depending on cellular conditions. Here we describe the behaviour of the univalent X chromosome in the wide-footed treehopper Enchenopa latipes (Say 1824). We confirm the chromosome number and sex determination method for this species, and that males possess a univalent X chromosome. We show that the univalent X chromosome forms a bipolar attachment to the spindle in metaphase I, and then segregates intact toward one spindle pole in late anaphase I (long after autosomes have initiated poleward movement). Movement of the univalent toward one pole is associated with loss of microtubule connections toward the opposite spindle pole.

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Micromanipulation of Chromosomes in Insect Spermatocytes

Abstract: Materials Name Company Catalog Number Comments VWR micro cover glass VWR 48366 249 25 mm x 25 mm, no 1.5

Cited by 9 publications

References 26 publications

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

Chromosome number, sex determination, and meiotic chromosome behavior in the praying mantid Hierodula membranacea

Segregation of the univalent X chromosome in the wide-footed treehopper Enchenopa latipes (Say 1824)

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