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1.
J Cell Biol ; 217(8): 2647-2659, 2018 08 06.
Article in English | MEDLINE | ID: mdl-29907657

ABSTRACT

For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct mechanisms: (1) capture of astral microtubules nucleated at the centrosome by the chromosomes' kinetochores or (2) attachment of kinetochores to noncentrosomal microtubules with subsequent transport of the minus ends of these microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal microtubules appear in the immediate vicinity of the kinetochores. Initially, these microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end-directed kinetochore-associated kinesin CenpE.


Subject(s)
Kinetochores/metabolism , Microtubules/metabolism , Prometaphase , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/physiology , Humans , Kinetochores/ultrastructure , Mad2 Proteins/metabolism , Microscopy, Electron , Microtubules/ultrastructure
2.
J Cell Biol ; 212(3): 307-19, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26833787

ABSTRACT

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.


Subject(s)
Kinetochores/drug effects , Mitosis/drug effects , Paclitaxel/pharmacology , Retinal Pigment Epithelium/drug effects , Cell Line , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins , Elasticity , Kinetochores/metabolism , Kinetochores/ultrastructure , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Electron , Microscopy, Fluorescence , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Conformation , Recombinant Fusion Proteins/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/ultrastructure , Stress, Mechanical , Time Factors , Transfection
3.
Nat Cell Biol ; 17(9): 1134-44, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26258631

ABSTRACT

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.


Subject(s)
Kinetochores/ultrastructure , Spindle Apparatus/metabolism , Cell Line , Chromosomes, Human/metabolism , Humans , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints , Microtubules/metabolism , Protein Transport
4.
J Cell Biol ; 206(2): 231-43, 2014 Jul 21.
Article in English | MEDLINE | ID: mdl-25023516

ABSTRACT

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.


Subject(s)
Chromosome Segregation , Chromosomes/metabolism , Kinetochores/metabolism , Spindle Poles/metabolism , Anaphase , Animals , Cells, Cultured , Chromosomes/ultrastructure , Humans , Kinetochores/ultrastructure , Marsupialia , Metaphase , Microtubules/metabolism , Microtubules/physiology , Microtubules/ultrastructure , Spindle Poles/ultrastructure
5.
Curr Opin Cell Biol ; 24(1): 40-7, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22221609

ABSTRACT

Kinetochores are complex macromolecular assemblies that link chromosomes to the mitotic spindle, mediate forces for chromosome motion, and generate the checkpoint signal delaying anaphase onset until all chromosomes are incorporated into the spindle. Proper execution of these functions depends on precise interactions between kinetochores and microtubules. While the molecular composition of the kinetochore is well described, structural organization of this organelle at the molecular and atomic levels is just beginning to emerge. Recent structural studies across scales suggest that kinetochores should not be viewed as rigid static scaffolds. Instead, these organelles exhibit a surprising degree of flexibility that enables rapid adaptations to various types of interactions with the mitotic spindle.


Subject(s)
Kinetochores/metabolism , Spindle Apparatus/metabolism , Anaphase , Animals , Chromosomes/metabolism , Humans , Kinetochores/chemistry , Microtubules/metabolism , Spindle Apparatus/chemistry
6.
PLoS One ; 5(6): e10978, 2010 Jun 07.
Article in English | MEDLINE | ID: mdl-20539754

ABSTRACT

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40-100 nm diameter x500-3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12alpha mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN- mediated killing.


Subject(s)
Cryptococcus gattii/pathogenicity , Neutrophils/microbiology , Animals , Arabidopsis/microbiology , Cryptococcus gattii/physiology , Cryptococcus gattii/ultrastructure , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred BALB C , Microscopy, Electron, Scanning , Plant Leaves/microbiology , Virulence
7.
Proc Natl Acad Sci U S A ; 107(23): 10484-9, 2010 Jun 08.
Article in English | MEDLINE | ID: mdl-20483991

ABSTRACT

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.


Subject(s)
Chickens , Kinetochores , Animals , Cell Line , Chickens/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Mitosis , Models, Biological , Protein Folding
8.
Cell Mol Life Sci ; 67(13): 2163-72, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20336345

ABSTRACT

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or "kinetochore fibrils" have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules.


Subject(s)
Kinetochores/metabolism , Microtubules/metabolism , Cytoskeletal Proteins , Humans , Mitosis , Models, Biological , Nuclear Proteins/metabolism , Spindle Apparatus/physiology
9.
Curr Biol ; 19(12): 1012-8, 2009 Jun 23.
Article in English | MEDLINE | ID: mdl-19481460

ABSTRACT

The centrosome is the principal microtubule organizing center (MTOC) of animal cells. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.


Subject(s)
Cell Division/physiology , Centrioles , Microtubule-Associated Proteins/metabolism , Animals , Cell Cycle/physiology , Cell Line , Centrioles/metabolism , Centrioles/ultrastructure , Humans , Microtubule-Associated Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure
10.
Cell ; 137(4): 672-84, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19450515

ABSTRACT

Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.


Subject(s)
Kinetochores/chemistry , Kinetochores/metabolism , Microtubules/chemistry , Microtubules/metabolism , Cytoskeletal Proteins , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Metaphase , Microscopy, Fluorescence , Microtubule-Associated Proteins/metabolism , Nuclear Proteins
11.
Mol Biol Cell ; 20(9): 2371-80, 2009 May.
Article in English | MEDLINE | ID: mdl-19261808

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Centromere/metabolism , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Vertebrates/metabolism , Animals , Autoantigens/metabolism , Cell Line , Centromere/ultrastructure , Centromere Protein A , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Gene Silencing , Green Fluorescent Proteins/metabolism , Humans , Kinetochores/metabolism , Kinetochores/ultrastructure , Microtubules/ultrastructure , Mitosis , Recombinant Fusion Proteins/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure
12.
J Cell Biol ; 184(3): 355-6, 2009 Feb 09.
Article in English | MEDLINE | ID: mdl-19193624

ABSTRACT

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.


Subject(s)
Kinetochores/metabolism , Spindle Apparatus/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Centromere Protein A , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins , HeLa Cells , Humans , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
13.
Cell ; 135(6): 1039-52, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-19070575

ABSTRACT

Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.


Subject(s)
Centromere , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Kinetochores/metabolism , Amino Acid Sequence , Animals , Autoantigens/genetics , Autoantigens/metabolism , Centromere Protein A , Chickens , Chromosomal Proteins, Non-Histone/genetics , HeLa Cells , Histones/metabolism , Humans , Mutation , Nucleosomes/metabolism
14.
Curr Biol ; 18(22): 1778-84, 2008 Nov 25.
Article in English | MEDLINE | ID: mdl-19026543

ABSTRACT

Accurate chromosome segregation is dependent upon stable attachment of kinetochores to spindle microtubules during mitosis. A long-standing question is how kinetochores maintain stable attachment to the plus ends of dynamic microtubules that are continually growing and shortening. The Ndc80 complex is essential for persistent end-on kinetochore-microtubule attachment in cells [1, 2], but how the Ndc80 complex forms functional microtubule-binding sites remains unknown. We show that the 80 amino acid N-terminal unstructured "tail" of Hec1 is required for generating stable kinetochore-microtubule attachments. PtK1 cells depleted of endogenous Hec1 and rescued with Hec1-GFP fusion proteins deleted of the entire N terminus or the disordered N-terminal 80 amino acid tail domain fail to generate stable kinetochore-microtubule attachments. Mutation of nine amino acids within the Hec1 tail to reduce its positive charge also abolishes stable attachment. Furthermore, the mitotic checkpoint remains functional after deletion of the N-terminal 80 amino acid tail, but not after deletion of the N-terminal 207 amino acid region containing both the tail domain and a calponin homology (CH) domain. These results demonstrate that kinetochore-microtubule binding is dependent on electrostatic interactions mediated through the disordered N-terminal 80 amino acid tail domain and mitotic-checkpoint function is dependent on the CH domain of Hec1.


Subject(s)
Kinetochores/metabolism , Microtubules/metabolism , Nuclear Proteins/chemistry , Animals , Binding Sites , Cell Line , Green Fluorescent Proteins/analysis , Kinetochores/ultrastructure , Microtubules/ultrastructure , Mutation , Nuclear Proteins/metabolism , Nuclear Proteins/physiology , Potoroidae , Protein Structure, Tertiary , RNA Interference , Recombinant Fusion Proteins/analysis
16.
Nat Cell Biol ; 9(5): 516-22, 2007 May.
Article in English | MEDLINE | ID: mdl-17435749

ABSTRACT

Intricate interactions between kinetochores and microtubules are essential for the proper distribution of chromosomes during mitosis. A crucial long-standing question is how vertebrate kinetochores generate chromosome motion while maintaining attachments to the dynamic plus ends of the multiple kinetochore MTs (kMTs) in a kinetochore fibre. Here, we demonstrate that individual kMTs in PtK(1) cells are attached to the kinetochore outer plate by several fibres that either embed the microtubule plus-end tips in a radial mesh, or extend out from the outer plate to bind microtubule walls. The extended fibres also interact with the walls of nearby microtubules that are not part of the kinetochore fibre. These structural data, in combination with other recent reports, support a network model of kMT attachment wherein the fibrous network in the unbound outer plate, including the Hec1-Ndc80 complex, dissociates and rearranges to form kMT attachments.


Subject(s)
Epithelial Cells/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Mitosis/physiology , Animals , Binding Sites , Cell Line , Epithelial Cells/ultrastructure , Kinetochores/ultrastructure , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Models, Molecular , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation , Tomography, X-Ray Computed , Vertebrates
18.
Chromosoma ; 115(6): 469-80, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16909258

ABSTRACT

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40-50 nm thick. On average, each S2 kinetochore binds 11+/-2 MTs, in contrast to the 4-6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.


Subject(s)
Drosophila melanogaster/cytology , Drosophila melanogaster/ultrastructure , Kinetochores/ultrastructure , Animals , Cells, Cultured , Centromere Protein A , Centromere Protein B/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Painting , Chromosomes/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Fluorescent Antibody Technique , HeLa Cells , Histones/metabolism , Humans , Metaphase , Microscopy, Electron , Microtubules/metabolism , Models, Molecular , Spindle Apparatus/metabolism
19.
IEEE Trans Inf Technol Biomed ; 10(3): 608-17, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16871731

ABSTRACT

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.


Subject(s)
Algorithms , Artificial Intelligence , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Microtubules/ultrastructure , Pattern Recognition, Automated/methods , Tomography, X-Ray Computed/methods , Computer Simulation , Information Storage and Retrieval/methods , Models, Anatomic , Models, Biological , Reproducibility of Results , Sensitivity and Specificity
20.
IEEE Trans Image Process ; 15(7): 2035-48, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16830922

ABSTRACT

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.


Subject(s)
Algorithms , Artificial Intelligence , Image Interpretation, Computer-Assisted/methods , Kinetochores/ultrastructure , Microtubules/ultrastructure , Pattern Recognition, Automated/methods , Tomography, X-Ray Computed/methods , Cluster Analysis , Image Enhancement/methods , Information Storage and Retrieval/methods , Reproducibility of Results , Sensitivity and Specificity , Subtraction Technique
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