Elsevier

Methods

Volume 41, Issue 2, February 2007, Pages 177-189
Methods

Analysis of kinetochore assembly and function in Caenorhabditis elegans embryos and human cells

https://doi.org/10.1016/j.ymeth.2006.07.027Get rights and content

Abstract

All eukaryotes rely on multi-protein assemblies, called kinetochores, to direct the segregation of their chromosomes in mitosis. The list of known kinetochore components has been growing rapidly in the post-genomic era: in animal cells, there are presently more than 80 proteins that show either exclusive or partial localization at kinetochores during mitosis. The future challenge is to elucidate how these proteins contribute to kinetochore structure, spindle microtubule attachment, regulation of microtubule dynamics, and the detection, signaling, and correction of microtubule attachment errors. Cultured human tumor cells, especially HeLa cells, are widely used for the study of kinetochores. Recently, the experimental advantages offered by the nematode Caenorhabditis elegans have been exploited for functional analysis of kinetochore components in the first embryonic division. Here, we discuss basic methods, largely based on fluorescence imaging, to study kinetochore structure and function in these two metazoan model systems.

Introduction

The kinetochore is a complex proteinaceous structure that mediates and monitors the attachment of spindle microtubules to chromosomes and actively participates in chromosome alignment and the segregation of sister chromatids. It is built on a specialized chromatin foundation at the centromere, which is marked by the presence of the histone H3 variant CENP-A. Two types of chromosome architectures are prevalent in eukaryotes: monocentric, where localized kinetochores form at the primary constriction of sister chromatids, and holocentric, where kinetochores extend along the entire length of each chromatid. Despite this difference in chromosome architecture, the structural and functional properties of kinetochores are well conserved. Structural studies using electron microscopy [1], [2], [3] have led to the notion that the kinetochore consists of a chromatin–proximal or inner kinetochore domain that provides the foundation for building the kinetochore–microtubule interface or outer domain. In the absence of microtubules, an additional structure, termed the fibrous corona, is observed emanating from the outer domain into the cytoplasm. Each mammalian kinetochore binds between 20 and 40 microtubules, which form a bundle that is referred to as a kinetochore (K-) fiber [4]. Electron microscopy continues to make significant contributions to the structural understanding of kinetochores, especially at the kinetochore–microtubule interface [5]. Furthermore, immunoelectron microscopy has provided molecular landmarks for different layers of the kinetochore [6], [7], which in turn provide standards for analyzing the location of newly identified components within the substructure of the kinetochore using light microscopy.

The kinetochore is a dynamic structure whose composition is cell cycle-dependent [8], [9]. Most kinetochore components, for example the motor protein CENP-E or the outer plate constituent HEC1/Ndc80, assemble in prophase and the kinetochore becomes competent to engage spindle microtubules after nuclear envelope breakdown. These transient mitotic components leave the kinetochore at different times during mitosis and are absent in interphase. In contrast, constitutive components such as the inner kinetochore component CENP-A remain at the kinetochore throughout the cell cycle and ‘mark’ the kinetochore location (termed the pre-kinetochore) on the decondensed interphase chromatin.

The capture of spindle microtubules by mature kinetochores in prometaphase leads to the stochastic congression of each chromosome to the spindle equator. To avoid chromosome missegregation, the sister chromatids of every chromosome must be attached to K-fibers from opposite poles, a state referred to as bi-orientation or amphitelic attachment. A signaling network, called the spindle assembly checkpoint [10], [11], [12], generates a diffusible inhibitor at kinetochores of sister chromatids that have not yet achieved bi-orientation, delaying anaphase onset. The checkpoint is rapidly silenced once all kinetochores are properly attached.

Interfering with kinetochore function results in defects in chromosome alignment, sister chromatid segregation, and cell cycle progression. All of these processes are best studied using fluorescence imaging to visualize individual fixed and living cells, although population assays, such as flow cytometric analysis of DNA content, are also commonly utilized. Here, we focus on single-cell visual assays which have been essential for the study of kinetochore assembly and for interpreting consequences of inhibiting specific kinetochore proteins.

Section snippets

Indirect immunofluorescence

The optimal fixation condition for indirect immunofluorescence of kinetochore components is often a compromise between obtaining satisfactory staining for the kinetochore antigen and preserving the morphology of mitotic chromosomes and spindle microtubules. Glutaraldehyde fixation preserves cell ultrastructure best, especially spindle microtubules, but has an adverse effect on antigenicity. Formaldehyde fixation is less suitable for microtubules, but preserves condensed chromosome morphology

RNA interference

RNAi is the most popular technique to study function of genes required for essential subcellular processes in C. elegans. The chapters ‘Reverse Genetics’ and ‘Cell Division’ at www.wormbook.org offer a detailed description of the technique. Protein depletion is efficient (usually >90%) and largely independent of protein turnover. CENP-A, for example, is difficult to deplete in human cells, but is readily depleted in C. elegans [15]. Because the first embryonic division in the near-absence of a

Functional analysis of kinetochore assembly

Studies of localization dependencies between kinetochore components have revealed conserved mechanisms of kinetochore assembly. Work so far has found largely linear dependency relationships with the recruitment of outer kinetochore components depending on the presence of inner kinetochore components. CENP-A, which is incorporated into centromeric nucleosomes, is at the ‘top’ of the assembly hierarchy and components of the kinetochore–microtubule interface are at the ‘bottom.’ To place a

Fixed cells

For a first analysis of potential mitotic defects after a kinetochore component knockdown, cells are fixed and stained for tubulin, DNA, and a constitutive kinetochore marker (such as CREST serum for human cells). With these three markers, the cytologically defined mitotic phases are readily distinguished:

  • 1.

    Prophase: chromosomes condense within the confines of the nucleus.

  • 2.

    Prometaphase: chromosomes attach to microtubules and align on the bipolar spindle.

  • 3.

    Metaphase: all chromosomes are aligned at

Acknowledgments

Work in the Desai laboratory is supported by the LICR and a Grant from the NIH to A.D. (R01GM074215-01). R.G. is a Swiss National Science Foundation Fellow, S.K. is a Fellow of the American Cancer Society, and A.D. is the Connie and Bob Lurie Scholar of the Damon Runyon Cancer Research Foundation (DRS 38-04).

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