Reconstitution and Functional Analysis of Kinetochore Subcomplexes

Gestaut, Daniel R.; Cooper, Jeremy; Asbury, Charles L.; Davis, Trisha N.; Wordeman, Linda

doi:10.1016/s0091-679x(10)95032-2

Cited by 21 publications

(26 citation statements)

References 17 publications

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“…Custom instrumentation and flow cells were prepared as previously described (23,39). All protein complexes were purified via gel filtration as described under Protein Expression and Purification.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

Kudalkar

Scarborough

Umbreit

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore-microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubulebinding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex.

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Section: Methodsmentioning

confidence: 99%

“…24. Single-particle tracking and analysis were done using custom Labview (National Instruments) and Igor Pro (Wavemetrics) software as previously described (23,39). See SI Materials and Methods for additional details.…”

Section: Methodsmentioning

confidence: 99%

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

Kudalkar

Scarborough

Umbreit

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Image analysis programs custom written in LabView (see Gestaut et al 2010) Copies of our programs are available for free upon request.…”

Section: Methodsmentioning

confidence: 99%

Data Analysis for Total Internal Reflection Fluorescence Microscopy

Asbury

2016

Cold Spring Harb Protoc

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In the microscopes we use to analyze total internal reflection fluorescence (TIRF), the emitted fluorescence is split chromatically, using dichroic filters, into either two or three different colors (“channels”). In our two-color instrument, the green emission wavelengths (405–488 nm; for imaging green fluorescent protein [GFP]-tagged proteins) and far-red emission wavelengths (650–800 nm; for imaging Alexa-647-labeled microtubules) are projected onto the upper and lower halves, respectively, of a single camera. A single filter can be swapped to collect near-red wavelengths (561–640 nm; for imaging mCherry, or Alexa-568-labeled microtubules) instead of far-red. Our three-color instrument is very similar except that the green, near-red, and far-red color ranges are projected onto three separate cameras. In either case, the different colors can be imaged simultaneously. Typically, we collect images at 10 frames/sec for ~200 sec. We have developed a series of semiautomated image analysis programs, written in LabView, to obtain the brightness, residence time, and mobility of individual particles bound to single microtubules. The basic analysis steps are straightforward and could also be implemented using ImageJ or Matlab. For convenience, this protocol describes the analysis of a single microtubule. Data from many microtubules across many experimental trials are needed to obtain robust conclusions that are independent of stochastic and trial-to-trial variability.

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“…This protocol for utilizing TIRF microscopy to study the binding of kinetochore proteins to microtubules at the single-molecule level is adapted from Gestaut et al (2010).…”

Section: Methodsmentioning

confidence: 99%

Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy

Kudalkar

Davis

Asbury

2016

Cold Spring Harb Protoc

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Here we present our standard protocol for studying the binding of kinetochore proteins to microtubules as a paradigm for designing single-molecule total internal reflection fluorescence (TIRF) microscopy experiments. Several aspects of this protocol require empirical optimization, including the method for anchoring the polymer or substrate to the coverslip, the type and amount of blocking protein to prevent nonspecific protein adsorption to the glass, the appropriate protein concentration, the laser power, and the duration of imaging. Our method uses bovine serum albumin and κ-casein as blocking agents to coat any imperfections in the coverslip silanization and thereby prevent protein adsorption to the coverslip. Protein concentration and duration of imaging must be optimized for each experiment and protein of interest. Ideally, a range is determined that allows for resolution of single complexes binding to microtubules to ensure proper measurement of kinetic off rates and diffusion along microtubules. Excessively high concentrations may lead to overlapping binding of proteins on microtubules, making it impossible to resolve single binding events. The duration of imaging must be long enough to capture very low off rates (long residence time on microtubules) and we typically image at 10 frames/sec for 200 sec. The laser power can be adjusted to prevent photobleaching, but must be high enough to achieve a sufficient signal/noise ratio.

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Reconstitution and Functional Analysis of Kinetochore Subcomplexes

Cited by 21 publications

References 17 publications

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

Data Analysis for Total Internal Reflection Fluorescence Microscopy

Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy

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