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Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.

The nucleus is a mechanically stable compartment of the cell that contains the genome and performs many essential functions. Nuclear mechanical components chromatin and lamins maintain nuclear shape, compartmentalization, and function by resisting antagonistic actin contraction and confinement. However, studies have yet to compare chromatin and lamins perturbations side-by-side as well as modulated actin contraction while holding confinement constant. To accomplish this, we used NLS-GFP to measure nuclear shape and rupture in live cells with chromatin decompaction (VPA), loss of lamin B1 (LMNB1-/-), and loss of lamin A/C (LMNA-/-). We then modulated actin contraction while maintaining actin confinement measured by nuclear height. Wild type, chromatin decompaction, and lamin B1 null present bleb-based nuclear deformations and ruptures dependent on actin contraction and independent of actin confinement. Inhibition of actin contraction by Y27632 decreased nuclear blebbing and ruptures to near 0% of cells while activation of actin contraction by CN03 increased the frequency of ruptures by nearly two-fold. However, lamin A/C null results in overall abnormal shape, but similar blebs and ruptures as wild type which were unaffected by actin contraction modulation. Actin contraction control of nuclear shape and ruptures showed that DNA damage levels were more correlated with perturbed nuclear shape than they were with changes in nuclear ruptures. We reveal that lamin B1 is a chromatin decompaction phenotype because using GSK126, which mimics the loss of facultative heterochromatin in lamin B1 null, is sufficient to phenocopy increased nuclear blebbing and ruptures. Furthermore, even though blebs and ruptures in lamin A/C null cells are insensitive to actin contraction, they do have the capacity to form increased levels of nuclear blebs and bleb-based ruptures, shown by treating with VPA. Thus, nuclear bleb formation and bleb-based nuclear ruptures are driven by actin contraction and independent of changes in actin confinement.

Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. The biophysical properties of chromatin alter nuclear shape and stability, but little is known about whether or how major genomic functions can impact the integrity of the nucleus. We hypothesized that transcription might affect cell nuclear shape and rupture through its effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with the RNA polymerase II inhibitor alpha-amanitin in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription is necessary for robust nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. These morphological effects appear to occur through a novel biophysical pathway, since transcription does not alter either chromatin histone modification state or nuclear rigidity, which typically control nuclear blebbing. We find that active/phosphorylated RNA pol II Ser5, marking transcription initiation, is enriched in nuclear blebs relative to DNA. Thus, transcription initiation is a hallmark of nuclear blebs. Polymer simulations suggest that motor activity within chromatin, such as that of RNA pol II, can generate active forces that deform the nuclear periphery, and that nuclear deformations depend on motor dynamics. Our data provide evidence that the genomic function of transcription impacts nuclear shape stability, and suggests a novel mechanism, separate and distinct from chromatin rigidity, for regulating large-scale nuclear shape and function.

Intro Force measurements of the nucleus, the strongest organelle, have propelled the field of mechanobiology to understand the basic mechanical components of the nucleus and how these components properly support nuclear morphology and function. Micromanipulation force measurement provides separation of the relative roles of nuclear mechanical components chromatin and lamin A. Methods To provide access to this technique, we have developed a universal micromanipulation apparatus for inverted microscopes. We outline how to engineer and utilize this apparatus through dual micromanipulators, fashion and calibrate micropipettes, and flow systems to isolate a nucleus and provide force vs. extensions measurements. This force measurement approach provides the unique ability to measure the separate contributions of chromatin at short extensions and lamin A strain stiffening at long extensions. We then investigated the apparatus’ controllable and programmable micromanipulators through compression, isolation, and extension in conjunction with fluorescence to develop new assays for nuclear mechanobiology. Results Using this methodology, we provide the first rebuilding of the micromanipulation setup outside of its lab of origin and recapitulate many key findings including spring constant of the nucleus and strain stiffening across many cell types. Furthermore, we have developed new micromanipulation-based techniques to compress nuclei inducing nuclear deformation and/or rupture, track nuclear shape post-isolation, and fluorescence imaging during micromanipulation force measurements. Conclusion We provide the workflow to build and use a micromanipulation apparatus with any inverted microscope to perform nucleus isolation, force measurements, and various other biophysical techniques.

Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus–locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10–25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics. Liquid chromatin Hi-C maps the intrinsic stability of associations between loci. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic.

© 2020 Elsevier Inc. Nucleosomes package genomic DNA into chromatin. By regulating DNA access for transcription, replication, DNA repair, and epigenetic modification, chromatin forms the nexus of most nuclear processes. In addition, dynamic organization of chromatin underlies both regulation of gene expression and evolution of chromosomes into individualized sister objects, which can segregate cleanly to different daughter cells at anaphase. This collaborative review shines a spotlight on technologies that will be crucial to interrogate key questions in chromatin and chromosome biology including state-of-the-art microscopy techniques, tools to physically manipulate chromatin, single-cell methods to measure chromatin accessibility, computational imaging with neural networks and analytical tools to interpret chromatin structure and dynamics. In addition, this review provides perspectives on how these tools can be applied to specific research fields such as genome stability and developmental biology and to test concepts such as phase separation of chromatin.

As the home of the cell’s genetic information, the nucleus plays a critical role in determining the cell’s fate and function in response to various signals and stimuli. In addition to biochemical inputs, the nucleus is constantly exposed to intrinsic and extrinsic mechanical forces that trigger dynamic changes in nuclear structure and morphology. Emerging data suggest that the physical deformation of the nucleus modulates many cellular and nuclear functions, which have long been considered downstream of cytoplasmic signaling pathways and dictated by DNA genomic sequences. In this Review, we discuss an emerging perspective on the mechanoregulation of the genetic machinery that considers the physical connections from chromatin to nuclear lamina and cytoskeletal filaments as a single mechanical unit. We describe key mechanisms of the spatial and temporal coordination of nuclear deformations and provide a critical review of the structural and functional adaptive responses of the nucleus to deformations. We then consider the contribution of nuclear deformations to the regulation of important cellular functions, including muscle contraction, cell migration, and human disease pathogenesis. Collectively, these emerging insights shed new light on the dynamics of nuclear deformations and their roles in cellular mechanobiology.

The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.

Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells., Methods to track molecular motion in eukaryotic cells mostly rely on fluorescent labels, transfection or photobleaching. Here the authors use multimodal partial wave spectroscopy to perform label-free live cell measurements of nanoscale structure and macromolecular motion with millisecond temporal resolution.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165Eindicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Irregular nuclear shapes characterized by blebs, lobules, micronuclei, or invaginations are hallmarks of many cancers and human pathologies. Despite the correlation between abnormal nuclear shape and human pathologies, the mechanism by which the cancer nucleus becomes misshapen is not fully understood. Motivated by recent evidence that modifying chromatin condensation can change nuclear morphology, we conducted a high-throughput RNAi screen to identify epigenetic regulators that are required to maintain normal nuclear shape in human breast epithelial MCF-10A cells. We silenced 608 genes in parallel using an epigenetics siRNA library and used an unbiased Fourier analysis approach to quantify nuclear contour irregularity from fluorescent images captured on a high-content microscope. Using this quantitative approach, which we validated with confocal microscopy, we significantly expand the list of epigenetic regulators that impact nuclear morphology.

Nuclei are constantly under external stress – be it during migration through tight constrictions or compressive pressure by the actin cap – and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

The nucleus is the organelle in the cell that contains the genome and its associate proteins which is collectively called chromatin. New work has shown that chromatin and its compaction level, dictated largely through histone modification state, provides rigidity to protect and stabilize the nucleus. Alterations in chromatin, its mechanics, and downstream loss of nuclear shape and stability are hallmarks of human disease. Weakened nuclear mechanics and abnormal morphology have been shown to cause rupturing of the nucleus which results in nuclear dysfunction including DNA damage. Thus, the rigidity provided by chromatin to maintain nuclear mechanical stability also provides its own protection from DNA damage via compartmentalization maintenance.

The cell nucleus houses, protects, and arranges the genome within the cell. Therefore, nuclear mechanics and morphology are important for dictating gene regulation, and these properties are perturbed in many human diseases, such as cancers and progerias. The field of nuclear mechanics has long been dominated by studies of the nuclear lamina, the intermediate filament shell residing just beneath the nuclear membrane. However, a growing body of work shows that chromatin and chromatin-related factors within the nucleus are an essential part of the mechanical response of the cell nucleus to forces. Recently, our group demonstrated that chromatin and the lamina provide distinct mechanical contributions to nuclear mechanical response. The lamina is indeed important for robust response to large, whole-nucleus stresses, but chromatin dominates the short-extension response. These findings offer a clarifying perspective on varied nuclear mechanics measurements and observations, and they suggest several new exciting possibilities for understanding nuclear morphology, organization, and mechanics.

We study a Brownian dynamics simulation model of a biopolymeric shell deformed by axial forces exerted at opposing poles. The model exhibits two distinct linear force-extension regimes, with the response to small tensions governed by linear elasticity and the response to large tensions governed by an effective spring constant that scales with radius as R−0.25. When extended beyond the initial linear elastic regime, the shell undergoes a hysteretic, temperature-dependent buckling transition. We experimentally observe this buckling transition by stretching and imaging the lamina of isolated cell nuclei. Furthermore, the interior contents of the shell can alter mechanical response and buckling, which we show by simulating a model for the nucleus that quantitatively agrees with our micromanipulation experiments stretching individual nuclei.

Micromanipulation force measurements of single isolated nuclei at physiological strains and strain rates reveal two distinct cell nuclear mechanical regimes differentially governed by chromatin and lamin A. Chromatin and its histone-modification compaction govern short extension, and the lamin A amount dictates long-extension strain stiffening., The cell nucleus must continually resist and respond to intercellular and intracellular mechanical forces to transduce mechanical signals and maintain proper genome organization and expression. Altered nuclear mechanics is associated with many human diseases, including heart disease, progeria, and cancer. Chromatin and nuclear envelope A-type lamin proteins are known to be key nuclear mechanical components perturbed in these diseases, but their distinct mechanical contributions are not known. Here we directly establish the separate roles of chromatin and lamin A/C and show that they determine two distinct mechanical regimes via micromanipulation of single isolated nuclei. Chromatin governs response to small extensions (

SUMMARYChromosomes are folded so that active and inactive chromatin domains are spatially segregated. Compartmentalization is thought to occur through polymer phase/microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented removing the strong polymeric constraint to enable detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are over 10-25 kb. Fragmenting chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Dissolution kinetics of chromatin interactions vary for different chromatin domains. Lamin-associated domains are most stable, while interactions among speckle and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation, possibly because cohesin rings slide off nearby DNA ends. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.HighlightsLiquid chromatin Hi-C detects chromatin interaction dissociation rates genome-wideChromatin conformations in distinct nuclear compartments differ in stabilityStable heterochromatic associations are major drivers of chromatin phase separationCTCF-CTCF loops are stabilized by encirclement of loop bases by cohesin rings

The cell nucleus encloses, organizes, and protects the genome. Chromatin maintains nuclear mechanical stability and shape in coordination with lamins and the cytoskeleton. Abnormal nuclear shape is a diagnostic marker for human diseases, and it can cause nuclear dysfunction. Chromatin mechanics underlies this link, as alterations to chromatin and its physical properties can disrupt or rescue nuclear shape. The cell can regulate nuclear shape through mechanotransduction pathways that sense and respond to extracellular cues, thus modulating chromatin compaction and rigidity. These findings reveal how chromatin's physical properties can regulate cellular function and drive abnormal nuclear morphology and dysfunction in disease.

We present a multimodal label-free interferometric imaging platform for measuring intracellular nanoscale structure and macromolecular dynamics in living cells with a sensitivity to macromolecules as small as 20nm and millisecond temporal resolution. We validate this system by pairing experimental measurements of nanosphere phantoms with a novel interferometric theory. Applying this system in vitro, we explore changes in higher-order chromatin structure and dynamics that occur due to cellular fixation, stem cell differentiation, and ultraviolet (UV) light irradiation. Finally, we discover a new phenomenon, cellular paroxysm, a near-instantaneous, synchronous burst of motion that occurs early in the process of UV induced cell death. Given this platform’s ability to obtain nanoscale sensitive, millisecond resolved information within live cells without concerns of photobleaching, it has the potential to answer a broad range of critical biological questions about macromolecular behavior in live cells, particularly about the relationship between cellular structure and function.

The pericentromere chromatin protrudes orthogonally from the sister-sister chromosome arm axis. Pericentric protrusions are organized in a series of loops with the centromere at the apex, maximizing its ability to interact with stochastically growing and shortening kinetochore microtubules. Each pericentromere loop is ∼50 kb in size and is organized further into secondary loops that are displaced from the primary spindle axis. Cohesin and condensin are integral to mechanisms of loop formation and generating resistance to outward forces from kinesin motors and anti-parallel spindle microtubules. A major unanswered question is how the boundary between chromosome arms and the pericentromere is established and maintained. We used sister chromatid separation and dynamics of LacO arrays distal to the pericentromere to address this issue. Perturbation of chromatin spring components results in 2 distinct phenotypes. In cohesin and condensin mutants sister pericentric LacO arrays separate a defined distance independent of spindle length. In the absence of Smt4, a peptidase that removes SUMO modifications from proteins, pericentric LacO arrays separate in proportion to spindle length increase. Deletion of Smt4, unlike depletion of cohesin and condensin, causes stretching of both proximal and distal pericentromere LacO arrays. The data suggest that the sumoylation state of chromatin topology adjusters, including cohesin, condensin, and topoisomerase II in the pericentromere, contribute to chromatin spring properties as well as the sister cohesion boundary.

Chromatin decompaction via increasing euchromatin or decreasing heterochromatin results in a softer nucleus and abnormal nuclear blebbing, independent of lamin perturbations. Conversely, increasing heterochromatin stiffens the nucleus and rescues nuclear morphology in lamin-perturbed cells that present abnormal nuclear morphology., Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed “blebs” are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson–Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity.

One of the most fundamental concepts of microscopy is that of resolution-the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope-deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy.

SummaryThe budding yeast kinetochore is ∼68 nm in length with a diameter slightly larger than a 25 nm microtubule [1]. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules [2]. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps [2] to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.

During mitosis, cohesin and condensin cross-link pericentromeres of different chromosomes to coordinate centromere attachment sites., The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.

The mitotic chromatin spring is organized into a rosette of intramolecular loops of pericentric chromatin by condensin and cohesin. Model convolution reveals that condensin clusters along the spindle axis, while cohesin is dispersed radially along pericentromere loops., In mitosis, the pericentromere is organized into a spring composed of cohesin, condensin, and a rosette of intramolecular chromatin loops. Cohesin and condensin are enriched in the pericentromere, with spatially distinct patterns of localization. Using model convolution of computer simulations, we deduce the mechanistic consequences of their spatial segregation. Condensin lies proximal to the spindle axis, whereas cohesin is radially displaced from condensin and the interpolar microtubules. The histone deacetylase Sir2 is responsible for the axial position of condensin, while the radial displacement of chromatin loops dictates the position of cohesin. The heterogeneity in distribution of condensin is most accurately modeled by clusters along the spindle axis. In contrast, cohesin is evenly distributed (barrel of 500-nm width × 550-nm length). Models of cohesin gradients that decay from the centromere or sister cohesin axis, as previously suggested, do not match experimental images. The fine structures of cohesin and condensin deduced with subpixel localization accuracy reveal critical features of how these complexes mold pericentric chromatin into a functional spring.

Dynamics of histones under tension in the pericentromere depends on RSC and ISW2 chromatin remodeling. The underlying pericentromeric chromatin forms a platform that is required to maintain kinetochore structure when under spindle-based tension., Nucleosome positioning is important for the structural integrity of chromosomes. During metaphase the mitotic spindle exerts physical force on pericentromeric chromatin. The cell must adjust the pericentromeric chromatin to accommodate the changing tension resulting from microtubule dynamics to maintain a stable metaphase spindle. Here we examine the effects of spindle-based tension on nucleosome dynamics by measuring the histone turnover of the chromosome arm and the pericentromere during metaphase in the budding yeast Saccharomyces cerevisiae. We find that both histones H2B and H4 exhibit greater turnover in the pericentromere during metaphase. Loss of spindle-based tension by treatment with the microtubule-depolymerizing drug nocodazole or compromising kinetochore function results in reduced histone turnover in the pericentromere. Pericentromeric histone dynamics are influenced by the chromatin-remodeling activities of STH1/NPS1 and ISW2. Sth1p is the ATPase component of the Remodels the Structure of Chromatin (RSC) complex, and Isw2p is an ATP-dependent DNA translocase member of the Imitation Switch (ISWI) subfamily of chromatin-remodeling factors. The balance between displacement and insertion of pericentromeric histones provides a mechanism to accommodate spindle-based tension while maintaining proper chromatin packaging during mitosis.

Summary Background Tension sensing of bioriented chromosomes is essential for the fidelity of chromosome segregation. The spindle assembly checkpoint (SAC) conveys lack of tension or attachment to the anaphase promoting complex. Components of the SAC (Bub1) phosphorylate histone H2A (S121) and recruit the protector of cohesin, Shugoshin (Sgo1), to the inner centromere. How the chromatin structural modifications of the inner centromere are integrated into the tension sensing mechanisms and the checkpoint are not known. Results We have identified a Bub1/Sgo1-dependent structural change in the geometry and dynamics of kinetochores and the pericentric chromatin upon reduction of microtubule dynamics. The cluster of inner kinetochores contract, whereas the pericentric chromatin and cohesin that encircle spindle microtubules undergo a radial expansion. Despite its increased spatial distribution, the pericentric chromatin is less dynamic. The change in dynamics is due to histone H2A phosphorylation and Sgo1 recruitment to the pericentric chromatin, rather than microtubule dynamics. Conclusions Bub1 and Sgo1 act as a rheostat to regulate the chromatin spring and maintain force balance. Through histone H2A S121 phosphorylation and recruitment of Sgo1, Bub1 kinase softens the chromatin spring in response to changes in microtubule dynamics. The geometric alteration of all 16 kinetochores and pericentric chromatin reflect global changes in the pericentromeric region and provide mechanisms for mechanically amplifying damage at a single kinetochore microtubule.

Pericentric enrichment of condensin on budding yeast chromosomes, which contributes to chromatin compaction and mitotic spindle structure and integrity, is mediated by condensin interaction with tRNA genes and the tRNA-interacting protein dyskerin., Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

During mitosis, spindle microtubule force is balanced by the combined activities of the cohesin and condensin SMC complexes and intramolecular pericentric chromatin loops., Sister chromatid cohesion provides the mechanistic basis, together with spindle microtubules, for generating tension between bioriented chromosomes in metaphase. Pericentric chromatin forms an intramolecular loop that protrudes bidirectionally from the sister chromatid axis. The centromere lies on the surface of the chromosome at the apex of each loop. The cohesin and condensin structural maintenance of chromosomes (SMC) protein complexes are concentrated within the pericentric chromatin, but whether they contribute to tension-generating mechanisms is not known. To understand how pericentric chromatin is packaged and resists tension, we map the position of cohesin (SMC3), condensin (SMC4), and pericentric LacO arrays within the spindle. Condensin lies proximal to the spindle axis and is responsible for axial compaction of pericentric chromatin. Cohesin is radially displaced from the spindle axis and confines pericentric chromatin. Pericentric cohesin and condensin contribute to spindle length regulation and dynamics in metaphase. Together with the intramolecular centromere loop, these SMC complexes constitute a molecular spring that balances spindle microtubule force in metaphase.

We previously described the Trypanin family of cytoskeleton-associated proteins that have been implicated in dynein regulation [Hill et al., J Biol Chem2000; 275(50):39369–39378; Hutchings et al., J Cell Biol2002;156(5):867–877; Rupp and Porter, J Cell Biol2003;162(1):47–57]. Trypanin from T. brucei is part of an evolutionarily conserved dynein regulatory system that is required for regulation of flagellar beat. In C. reinhardtii, the trypanin homologue (PF2) is part of an axonemal ‘dynein regulatory complex’ (DRC) that functions as a reversible inhibitor of axonemal dynein [Piperno et al., J Cell Biol1992;118(6):1455–1463; Gardner et al., J Cell Biol1994;127(5):1311–1325]. The DRC consists of an estimated seven polypeptides that are tightly associated with axonemal microtubules. Association with the axoneme is critical for DRC function, but the mechanism by which it attaches to the microtubule lattice is completely unknown. We demonstrate that Gas11, the mammalian trypanin/PF2 homologue, associates with microtubules in vitro and in vivo. Deletion analyses identified a novel microtubule-binding domain (GMAD) and a distinct region (IMAD) that attenuates Gas11-microtubule interactions. Using single-particle binding assays, we demonstrate that Gas11 directly binds microtubules and that the IMAD attenuates the interaction between GMAD and the microtubule. IMAD is able to function in either a cis- or trans-orientation with GMAD. The discovery that Gas11 provides a direct linkage to microtubules provides new mechanistic insight into the structural features of the dynein-regulatory complex. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc.

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.