Biology subjects

Biggs, R. J.

Publications and source records attributed to Biggs, R. J..

5 recordsLinked to original sources

bioRxiv2026

TMEM209/NPP-28 partitions PLK-1 between the nuclear envelope and nuclear interior during mitosis

The onset of mitosis in animal cells is marked by nuclear envelope breakdown (NEBD), which requires coordinated disassembly of nuclear pore complexes (NPCs) and the nuclear lamina. Polo-like kinase 1 (Plk1/PLK-1) initiates NEBD through phosphorylation of NPC components, but mechanisms that spatially regulate PLK-1 between NPCs and the nuclear interior are unclear. Here, we identify the Caenorhabditis elegans TMEM209 homolog NPP-28 as a transmembrane nucleoporin that concentrates PLK-1 at the nuclear envelope while limiting its nuclear accumulation to promote NEBD. We identify docking sites within NPP-28 that mediate PLK-1 nuclear envelope-association. NPP-28 is phosphorylated by PLK-1 in vitro, and mutational analysis suggests that NPP-28-mediated docking facilitates NEBD, whereas phosphorylation prevents premature NEBD. Simultaneous loss of npp-28 and the transmembrane nucleoporin npp-12/gp210 exacerbates mitotic NEBD defects in early embryos and causes severe defects in germline development. Together, our findings identify NPP-28 as a spatial regulator of PLK-1 during mitosis with functional relationships with other conserved transmembrane nucleoporins depending on context.

cell biology↗

bioRxiv2026

Mitotic chromosomes are mechanically connected by chromatin-based interchromosome linkers

The spatial organization of chromosomes is crucial for gene regulation and genome stability. Metaphase chromosomes appear physically discrete from one another in conventional chromosome preparations. However, interchromosomal connections, or "linkers", have been observed among mitotic chromosomes, and may play important roles in coordinated chromosome movements and genome stability. We use micropipette-based isolation and manipulation to analyze interchromosome linkers between mammalian mitotic chromosomes. Pulling an isolated chromosome reveals that thin linkers connect them; CREST staining indicates their locations to be at centromeres. The linkers display linear elasticity and have a length-doubling force of approximately 300 pN, comparable to the length-doubling force of an entire metaphase chromosome. Enzymatic treatments demonstrate that the linkers are disrupted by DNase, but not by RNase, protease, or microtubule polymerization (spindle fiber) inhibitors, indicating that linker connectivity is based on DNA. Immunofluorescence experiments indicate that histones and topo II are on the linkers, with topo II organized into discrete foci spaced by approximately 0.4 Mbp. After prolonged metaphase arrest using spindle inhibitors isolated genomes do not display interchromosome linkers. Finally, experiments on intact cells show that interchromosome linkers containing CENP-B can be observed between mitotic chromosomes, indicating they are not an artifact of genome isolation.

genomics↗

bioRxiv2024

Independence of centromeric and pericentromeric chromatin stability on CCAN components

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin and bound by the sequence specific binding protein CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromeres stiffness and the role of the CCAN. We degraded CENP-C and CENP-N using auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching approximately 3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.

cell biology↗

bioRxiv2020

HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics

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 HP1 (CBX5) is unknown. We used a novel HP1 auxin-inducible degron human cell line to rapidly degrade HP1. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1 is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1I165E indicate that chromatin crosslinking via HP1 dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1 similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1 is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

biophysics↗

bioRxiv2020

Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.

cell biology↗