Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
The key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture.
more » « less- NSF-PAR ID:
- 10393438
- Publisher / Repository:
- DOI PREFIX: 10.1083
- Date Published:
- Journal Name:
- Journal of Cell Biology
- Volume:
- 222
- Issue:
- 4
- ISSN:
- 0021-9525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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INTRODUCTION To faithfully distribute genetic material to daughter cells during cell division, spindle fibers must couple to DNA by means of a structure called the kinetochore, which assembles at each chromosome’s centromere. Human centromeres are located within large arrays of tandemly repeated DNA sequences known as alpha satellite (αSat), which often span millions of base pairs on each chromosome. Arrays of αSat are frequently surrounded by other types of tandem satellite repeats, which have poorly understood functions, along with nonrepetitive sequences, including transcribed genes. Previous genome sequencing efforts have been unable to generate complete assemblies of satellite-rich regions because of their scale and repetitive nature, limiting the ability to study their organization, variation, and function. RATIONALE Pericentromeric and centromeric (peri/centromeric) satellite DNA sequences have remained almost entirely missing from the assembled human reference genome for the past 20 years. Using a complete, telomere-to-telomere (T2T) assembly of a human genome, we developed and deployed tailored computational approaches to reveal the organization and evolutionary patterns of these satellite arrays at both large and small length scales. We also performed experiments to map precisely which αSat repeats interact with kinetochore proteins. Last, we compared peri/centromeric regions among multiple individuals to understand how these sequences vary across diverse genetic backgrounds. RESULTS Satellite repeats constitute 6.2% of the T2T-CHM13 genome assembly, with αSat representing the single largest component (2.8% of the genome). By studying the sequence relationships of αSat repeats in detail across each centromere, we found genome-wide evidence that human centromeres evolve through “layered expansions.” Specifically, distinct repetitive variants arise within each centromeric region and expand through mechanisms that resemble successive tandem duplications, whereas older flanking sequences shrink and diverge over time. We also revealed that the most recently expanded repeats within each αSat array are more likely to interact with the inner kinetochore protein Centromere Protein A (CENP-A), which coincides with regions of reduced CpG methylation. This suggests a strong relationship between local satellite repeat expansion, kinetochore positioning, and DNA hypomethylation. Furthermore, we uncovered large and unexpected structural rearrangements that affect multiple satellite repeat types, including active centromeric αSat arrays. Last, by comparing sequence information from nearly 1600 individuals’ X chromosomes, we observed that individuals with recent African ancestry possess the greatest genetic diversity in the region surrounding the centromere, which sometimes contains a predominantly African αSat sequence variant. CONCLUSION The genetic and epigenetic properties of centromeres are closely interwoven through evolution. These findings raise important questions about the specific molecular mechanisms responsible for the relationship between inner kinetochore proteins, DNA hypomethylation, and layered αSat expansions. Even more questions remain about the function and evolution of non-αSat repeats. To begin answering these questions, we have produced a comprehensive encyclopedia of peri/centromeric sequences in a human genome, and we demonstrated how these regions can be studied with modern genomic tools. Our work also illuminates the rich genetic variation hidden within these formerly missing regions of the genome, which may contribute to health and disease. This unexplored variation underlines the need for more T2T human genome assemblies from genetically diverse individuals. Gapless assemblies illuminate centromere evolution. ( Top ) The organization of peri/centromeric satellite repeats. ( Bottom left ) A schematic portraying (i) evidence for centromere evolution through layered expansions and (ii) the localization of inner-kinetochore proteins in the youngest, most recently expanded repeats, which coincide with a region of DNA hypomethylation. ( Bottom right ) An illustration of the global distribution of chrX centromere haplotypes, showing increased diversity in populations with recent African ancestry.more » « less
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