Bruce F. McEwen

The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547–1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous “precentrioles” become morphologically recognizable centrioles before mitosis. De novo–assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo–formed centrioles do not mature if they are assembled in S phase–arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.

A super-resolution map of the vertebrate kinetochore

A longstanding question in centromere biology has been the organization of CENP-A–containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A–rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than sur-rounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A–rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C–dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.

Condensin Regulates the Stiffness of Vertebrate Centromeres

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.

Alignment by Cross-Correlation

Highest precision in the alignment of projections is one of the prerequisites of tomography. The effects of random translational alignment errors on the reconstruction can be appreciated by considering the much simpler situation where a given number of images containing the same motif are averaged.

Adaptive changes in the kinetochore architecture facilitate proper spindle assembly

Mitotic spindle formation relies on the stochastic capture of microtubules at kinetochores. Kinetochore architecture affects the efficiency and fidelity of this process with large kinetochores expected to accelerate assembly at the expense of accuracy, and smaller kinetochores to suppress errors at the expense of efficiency. We demonstrate that on mitotic entry, kinetochores in cultured human cells form large crescents that subsequently compact into discrete structures on opposite sides of the centromere. This compaction occurs only after the formation of end-on microtubule attachments. Live-cell microscopy reveals that centromere rotation mediated by lateral kinetochore–microtubule interactions precedes the formation of end-on attachments and kinetochore compaction. Computational analyses of kinetochore expansion–compaction in the context of lateral interactions correctly predict experimentally observed spindle assembly times with reasonable error rates. The computational model suggests that larger kinetochores reduce both errors and assembly times, which can explain the robustness of spindle assembly and the functional significance of enlarged kinetochores.

A 360° single-axis tilt stage for the high-voltage electron microscope

A new type of specimen stage that permits more than 180° of tilting about the axis of a side‐entry rod has been developed for a high‐voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1‐MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single‐axis tomography, because it permits a complete 180° set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within ±0.20°, with a cumulative error of less than 1.0° over a 180° span.

Model-Based Automated Extraction of Microtubules From Electron Tomography Volume

We propose a model-based automated approach to extracting microtubules from noisy electron tomography volume. Our approach consists of volume enhancement, microtubule localization, and boundary segmentation to exploit the unique geometric and photometric properties of microtubules. The enhancement starts with an anisotropic invariant wavelet transform to enhance the microtubules globally, followed by a three-dimensional (3-D) tube-enhancing filter based on Weingarten matrix to further accentuate the tubular structures locally. The enhancement ends with a modified coherence-enhancing diffusion to complete the interruptions along the microtubules. The microtubules are then localized with a centerline extraction algorithm adapted for tubular objects. To perform segmentation, we novelly modify and extend active shape model method. We first use 3-D local surface enhancement to characterize the microtubule boundary and improve shape searching by relating the boundary strength with the weight matrix of the searching error. We then integrate the active shape model with Kalman filtering to utilize the longitudinal smoothness along the microtubules. The segmentation improved in this way is robust against missing boundaries and outliers that are often present in the tomography volume. Experimental results demonstrate that our automated method produces results close to those by manual process and uses only a fraction of the time of the latter

Automated extraction of fine features of kinetochore microtubules and plus-ends from electron tomography volume

Kinetochore microtubules (KMTs) and the associated plus-ends have been areas of intense investigation in both cell biology and molecular medicine. Though electron tomography opens up new possibilities in understanding their function by imaging their high-resolution structures, the interpretation of the acquired data remains an obstacle because of the complex and cluttered cellular environment. As a result, practical segmentation of the electron tomography data has been dominated by manual operation, which is time consuming and subjective. In this paper, we propose a model-based automated approach to extracting KMTs and the associated plus-ends with a coarse-to-fine scale scheme consisting of volume preprocessing, microtubule segmentation and plus-end tracing. In volume preprocessing, we first apply an anisotropic invariant wavelet transform and a tube-enhancing filter to enhance the microtubules at coarse level for localization. This is followed with a surface-enhancing filter to accentuate the fine microtubule boundary features. The microtubule body is then segmented using a modified active shape model method. Starting from the segmented microtubule body, the plus-ends are extracted with a probabilistic tracing method improved with rectangular window based feature detection and the integration of multiple cues. Experimental results demonstrate that our automated method produces results comparable to manual segmentation but using only a fraction of the manual segmentation time.

Releasing the spindle assembly checkpoint without tension

Eukaryotic cells have evolved a spindle assembly checkpoint (SAC) that facilitates accurate genomic segregation during mitosis by delaying anaphase onset in response to errors in kinetochore microtubule attachment. In contrast to the well-studied molecular mechanism by which the SAC blocks anaphase onset, the events triggering SAC release are poorly understood. Papers in this issue by Uchida et al. (Uchida, K.S.K., K. Takagaki, K. Kumada, Y. Hirayama, T. Noda, and T. Hirota. 2009. J. Cell Biol. 184:383–390) and Maresca and Salmon (Maresca, T.J., and E.D. Salmon. 2009. J. Cell Biol. 184:373–381) make an important advance by demonstrating that SAC release depends on molecular rearrangements within the kinetochore rather than tension-produced stretch between sister kinetochores.

Enhancement of microtubules in EM tomography

The interpretation of the EM tomography of microtubules is challenging due to the low SNR and low contrast of the volume data. Therefore, image enhancement is crucial for the subsequent segmentation and structural analysis of microtubules. In this paper, we propose a model based 3D image enhancement approach by combining transform domain technique and spatial domain techniques in three consecutive steps. The enhancement starts with an anisotropic invariant wavelet transform to effectively enhance the elongated features, followed by a 3D shape filter via eigen analysis to capture the local geometric properties of the tubular structure. The enhancement ends with a coherence enhancing diffusion to complete the interruptions along the microtubules. The contribution of this work is that we have tailored and improved each of the above techniques to exploit the unique geometric and photometric properties of microtubules. Experimental results indicate that our proposed approach has excellent performance in noise removal and enhancement of the tomography volume.