Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, ...called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.
Eukaryotic cells must first compact their chromosomes before faithfully segregating them during cell division. Failure to do so can lead to segregation defects with pathological consequences, such as ...aneuploidy and cancer. Duplicated interphase chromosomes are, therefore, reorganized into tight rods before being separated and directed to the newly forming daughter cells. This vital reorganization of chromatin remains poorly understood. To address the dynamics of mitotic condensation of single chromosomes in intact cells, we developed quantitative assays based on confocal time-lapse microscopy of live mammalian cells stably expressing fluorescently tagged core histones. Surprisingly, maximal compaction was not reached in metaphase, but in late anaphase, after sister chromatid segregation. We show that anaphase compaction proceeds by a mechanism of axial shortening of the chromatid arms from telomere to centromere. Chromatid axial shortening was not affected in condensin-depleted cells, but depended instead on dynamic microtubules and Aurora kinase. Acute perturbation of this compaction resulted in failure to rescue segregation defects and in multilobed daughter nuclei, suggesting functions in chromosome segregation and nuclear architecture.
The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by ...cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.
RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by ...microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.
Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the ...quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.
Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. ...At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2-BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly.
During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of ...mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.
Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by ...WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2‐7 subcomplex of the replicative Cdc45‐MCM‐GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL. Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.
Synopsis
Binding of the DNA replication helicase MCM to ESCO2 enables the acetyltransferase to acetylate cohesin complexes on nascent DNA. This modification protects cohesin from precocious release by WAPL and is required for maintenance of sister chromatid cohesion at centromeres.
ESCO2 is recruited to chromatin by MCM.
ESCO2‐MCM interactions are important for cohesin acetylation on nascent chromatin and centromeric cohesion.
ESCO1 and ESCO2 maintain cohesion between chromosome arms and centromeres, respectively.
Recruitment of cohesin acetyltransferase ESCO2 by the MCM2‐7 replication complex to chromatin is required for cohesin acetylation around replication forks to maintain centromeric cohesion.
Advances in imaging techniques and high-throughput technologies are providing scientists with unprecedented possibilities to visualize internal structures of cells, organs and organisms and to ...collect systematic image data characterizing genes and proteins on a large scale. To make the best use of these increasingly complex and large image data resources, the scientific community must be provided with methods to query, analyze and crosslink these resources to give an intuitive visual representation of the data. This review gives an overview of existing methods and tools for this purpose and highlights some of their limitations and challenges.
Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, ...we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome‐wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre‐mRNAs, including pre‐mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing “cohesion fatigue”. Simultaneous expression of sororin and APC2 from intron‐less cDNAs restores cohesion in SNW1‐depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome‐wide identification of retained introns in SNW1‐ and PRPF8‐depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.
Synopsis
Loss of splicesome subunits SNW1 and PRPF8 leads to altered intron retention in a specific set of genes and depletes expression of cohesion loading factor sororin, explaining the mitotic defects caused by aberrant pre‐mRNA splicing.
The spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion.
The main function of SNW1 in sister chromatid cohesion is to enable the splicing of pre‐mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2.
Defects in APC2 pre‐mRNA splicing reduce APC/C activity and contribute to cohesion defects by causing “cohesion fatigue”.
Transcriptome‐wide analysis reveal that a subset of splicing reactions is particularly sensitive to depletion of SNW1 or PRPF8.
The identification of introns that are retained in SNW1‐ or PRPF8‐depleted cells may help to understand phenotypes and diseases associated with splicing defects.
Loss of splicesome subunits SNW1 and PRPF8 leads to altered intron retention in a specific set of genes and depletes expression of cohesion loading factor sororin, explaining the mitotic defects caused by aberrant pre‐mRNA splicing.
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