Nucleus

The spatiotemporal organisation of chromatin in the eukaryotic nucleus is fundamental for genome regulation. Chromatin undergoes rapid remodelling and rearrangements within minutes, altering its diffusion properties. Considering the tight coupling between genome function and nuclear architecture, a key question is how chromatin dynamics adapt to or promote nuclear processes. To elucidate the underlying physical principles, we employed High-resolution Diffusion mapping (Hi-D) to track chromatin motion throughout interphase in live human cells. Our analysis, that considers both diffusive motion and drift generated by active forces, revealed that chromatin dynamics are heterogeneous, with distinct behaviours in different subnuclear zones. Notably, both diffusive and processive contributions to chromatin motion progressively decrease from G1 to G2 phase, with this reduction occurring uniformly across all subzones. This suggests a global mechanism driving the observed decrease in chromatin mobility during cell cycle progression. By combining genetic knockout experiments and polymer modelling, we demonstrate that the doubling of DNA content, rather than cohesin-mediated sister chromatid entrapment, is responsible for the gradual decrease in chromatin motion during the cell cycle in human nuclei. These findings provide new insights into the physical and functional organisation of chromatin and its regulation during cellular proliferation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712877v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@75c654org.highwire.dtl.DTLVardef@2fbd3dorg.highwire.dtl.DTLVardef@31025aorg.highwire.dtl.DTLVardef@191808e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Molecular crowding causes the compaction of chromatin fibers, contributing to the formation of the nuclear architecture. However, the molecular mechanism of compaction under crowded conditions is not yet fully understood. In this study, we employed the single-molecule optical tweezer method to investigate the effect of molecular crowding on chromatin structure. Force-extension experiments on a 12-mer polynucleosome in the presence of different sizes and concentrations of polyethylene glycol (PEG) as a crowding agent showed that at low concentrations of low-molecular-weight (MW) PEG, the compaction of the polynucleosome was not significant. In this respect, nucleosomes predominantly remained separated, while DNA-histone interactions within individual nucleosomes were slightly stabilized. In contrast, high concentrations of high-MW PEG significantly promote internucleosomal interactions, leading to highly compact polynucleosome conformations. Under these conditions, approximately 30 pN of force was required to disrupt the internucleosomal interactions and release DNA; this force was 36% higher than that required for DNA unwrapping in the absence of PEG. These findings suggest that molecular crowding impacts cellular processes by mechanically regulating chromatin accessibility for regulatory proteins and the passage of motor molecules such as RNA polymerase. Significance StatementChromatin condensation is closely related to biological processes such as transcription and replication. Molecular crowding has recently attracted attention as a factor regulating chromatin condensation. In this study, we used the optical tweezer method to analyze the molecular mechanisms underlying chromatin condensation. We found that high-molecular weight and high-concentration crowders (polyethylene glycol) induced significant compaction, which involved internucleosomal interactions that markedly reduced DNA accessibility. Our results suggest that molecular crowding not only alters the condensation state, but also mechanically regulates chromatin accessibility.

Nuclear blebs are herniations of the nucleus that occur in many human conditions including aging, heart disease, muscular dystrophy, and many cancers. Nuclear blebbing causes nuclear rupture and cellular dysfunction. However, understanding the formation, stability, and identification of nuclear blebs remains an ongoing challenge. Our previous studies reveal that nuclear blebs are best hallmarked by decreased DNA density. To determine if chromatin decompaction underlies decreased DNA density in nuclear blebs, we investigated the histone composition of nuclear blebs across multiple cell lines. Time lapse and immunofluorescence imaging revealed that global histone H2B and H3 levels are decreased in the nuclear bleb relative to the nuclear body. Next, we imaged histone modification states of euchromatin and heterochromatin, which respectively track decompact and compact states of chromatin. Overall, we find that nuclear blebs display variable histone modification state across cell lines, as euchromatin does not consistently enrich nor is heterochromatin consistently depleted. Nuclear blebs did consistently show active RNA Pol II initiation is enriched relative to elongation. Thus, we find that the local histone modification state is not an essential component of nuclear blebs while transcription initiation enrichment over elongation is reproducible across cell lines and conditions. Summary statementWe measured histones and their modification states in nuclear blebs. We find that chromatin state is variable while transcription initiation is consistently enriched relative to elongation in nuclear blebs.

Intron length is a fascinating example of form without function. The vast majority of intronic space within genomes remains without a provided utility. It often fascinates us to find introns performing any function at all, establishing an attention bias against the vast lacking of utility of the remaining intergenic space. In an attempt to better understand the greater breadth of intronic length, I investigate here what I term The Kinetic Intron Hypothesis. This hypothesis investigates hypothetical dynamics of intron RNA synthesis and degradation. It explores how NTPs stored within intron RNA might function in mitosis and NTP resource management. Preliminary testing of the hypothesis leads to trends that warrant further exploration and validation by the scientific community. SignificanceCurrently no widely acknowledged model exists to characterize the length of introns within genes, yet intron length is massively abundant in eukaryotic genomes. Here I present an attempt to model the length of introns. In doing so, I explore novel hypothesized intron dynamics, presenting preliminary data for previously uncharacterized intron characteristics. The new data and model have the protentional to unveil new avenues of utility for introns at the intracellular level.

In the sperm nucleus, protamine replaces histones to mediate extreme DNA compaction. The histone-to-protamine transition involves the occurrence of double-strand breaks, and is facilitated by transition proteins including those containing high-mobility-group (HMG) boxes. Here we used optical tweezers and microscopy to study the actions of HMGB1 and protamine on DNA. Confocal scans of GFP-HMGB1 on overstretched {lambda}-DNA show 2-3 foci that spread on the DNA upon retraction. Spreading of foci coincides with reannealing of ssDNA tracks, confirming their localization at ss-dsDNA junctions. Whereas the force-extension curves of protamine-bound {lambda}-DNA show tangles that withstand forces > 60 pN, premixing protamine with HMGB1 produces only bends and bridges ([~] 20 pN). The counteraction of HMGB1 involves its acidic C-terminal tail, as HMGB1-{Delta}C fails to prevent tangle formation. In line with these single-molecule results, brightfield and confocal imaging shows that HMGB1 converts protamine-dsDNA aggregates into liquid droplets whereas HMGB1-{Delta}C fails to do so. Together, these observations support our hypothesis that chromatin-associated proteins like HMGB1 help maintain early protamine-mediated DNA condensates in a liquid state, enabling the recruitment of the repair machinery to restore the duplex structure.

Chromosome segregation is a tightly-regulated process that normally occurs with high fidelity. Errors in chromosome segregation are associated with aging, cancer, and infertility. Initially erroneously attached chromosomes are corrected over the course of mitosis, with the spindle assembly checkpoint preventing entry into anaphase until this error correction is complete. Despite extensive work on the molecular basis of error correction and the spindle assembly checkpoint, it is still unclear how disruption of these processes contribute to chromosome segregation errors. Here, we develop and experimentally test a coarse-grained model of error correction in the presence of a faulty spindle assembly checkpoint. We use the resulting model to disentangle the impact of various small molecule and genetic perturbations on both error correction and the spindle assembly checkpoint, and to compare chromosomally stable hTERT-RPE-1 cells and chromosomally unstable U2-OS cells. We find that the probability of error-free chromosome segregation is determined by the ratio of the checkpoint failure rate to the error correction rate, and validate a simple heuristic for understanding the source of chromosome segregation errors: perturbations which cause errors by disrupting the spindle assembly checkpoint decrease anaphase times, while those that disrupt error correction increase anaphase times. Taken together, this work provides a quantitative framework for understanding how error correction and the spindle assembly checkpoint contribute to mitotic timing and fidelity.

1Nuclear envelope stretch and rupture are common to cell spreading and migration in a variety of microenvironments, leading to marked changes in nucleocytoplasmic transport. Predicting cell response to different mechanochemical cues that are transmitted to the nucleus remains an open problem in the field of mechanomedicine. We developed a predictive modeling framework to examine how nuclear deformation on substrates with different nanotopographies influences nucleocytoplasmic transport and rearrangement of the nuclear lamina. Using the finite element method, we simulated nuclear compression by the perinuclear actin cap on substrates with arrays of nanopillars, modeling the nuclear envelope as a nonlinear elastic structure and coupling deformations to a biochemical model of lamin remodeling and nucleocytoplasmic transport. These simulations predicted regions of high nuclear envelope stretch adjacent to cell-nanopillar contacts, leading to maximized nuclear envelope tension on small nanopillars spaced by 4-5 microns. We then considered the effects on nuclear transport of YAP and TAZ and found that increased nuclear compression led to YAP/TAZ nuclear localization in agreement with previous experiments. Furthermore, the simulated force load per lamin was maximized on nanopillar substrates with high nuclear stretch. The magnitude of this load was modulated by the rate of actin cap assembly and the overall expression level of lamin A/C - decreasing lamin content in the nuclear envelope led to a higher likelihood of rupture. We validated this prediction in subsequent experiments with lamin-depleted U2OS cells, establishing the central importance of lamin transport and microenvironment nanotopography to nuclear mechanotransduction. 2 SignificanceCell nuclei commonly experience large strains, but existing computational models do not explain the coupling between such deformations and molecular transport. Here, we present a modeling framework that includes the mechanics of nuclear deformations and the reaction-transport of molecules within the cytoplasm, nuclear envelope, and nuclear interior. As a well-controlled setup for comparing experiments and simulations, we consider nuclear indentations exhibited by cells on nanopillar substrates. Our simulations recapitulate measurements of nuclear YAP/TAZ localization from the literature and predict that low-lamin cells experience higher force loads at the nuclear envelope. We validate this prediction experimentally, showing that lamin-depleted cells are more likely to exhibit nuclear rupture. Overall, our framework presents opportunities to predict nuclear mechanoadaptation to different microenvironments.

Following DNA replication, cohesion maintains sister chromatids in spatial proximity with a certain degree of alignment. This tethering, mediated by the cohesin complex,may facilitate DNA repair and enable proper chromosome individualization and segregation during mitosis. However, it is still unclear how cohesion is established and how it reshapes the relative organization of replicated chromosomes to achieve its functions. In this study, we address these questions in the biological context of budding yeast, by disentangling the interplay between two major structural functions of cohesin: organizing individual chromatids through loop extrusion and sister chromatids through cohesion. Combining polymer modeling and detailed analysis of recent experimental data of replicated chromosomes in G2/M, we show that extruding and cohesive cohesins are sparsely distributed leading to mildly compacted and loosely aligned sister chromatids. Genome-wide analysis of inter-chromatid contact maps in WT and mutant conditions suggests that cohesion is asymmetric, favoring the tethering between non-homologous cohesin-enriched regions. Our work highlights the dual role played by cohesin in structuring the replicated genome and questions how homologous recombination may function in the context of asymmetric, partial alignment of sister chromatids. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/700293v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@112ae6org.highwire.dtl.DTLVardef@1176c54org.highwire.dtl.DTLVardef@c939d4org.highwire.dtl.DTLVardef@f390c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

The bacterial nucleoid undergoes extensive structural reorganization during growth, influenced by nucleoid-associated proteins (NAPs) whose interactions and effects on nucleoid organization remain unclear. We investigated these interactions by tracking single molecules of the NAP HU-PAmCherry in living Escherichia coli cells in different growth phases, and we further examined how two NAPs, Dps and H-NS, impact HU dynamics. HU mobility varies with growth phase: In exponential phase, HU has two distinct mobility states: a fast-diffusing state and a slower, interacting state. In stationary phase, we observed a third population of very slow molecules, suggesting stable HU binding or confinement within compacted DNA. Deleting dps increases HU mobility in stationary phase, consistent with findings that Dps promotes short-range DNA contacts and nucleoid compaction in deep stationary phase. We measured in exponential phase that hns deletion leads to nucleoid compaction, faster HU diffusion, and a third population of very slow HU molecules in these cells. In stationary phase, deleting hns increases these stably bound HU molecules. Our results show that growth-phase-dependent nucleoid reorganization by Dps and H-NS influences the behavior and function of other NAPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/695591v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@16d8f9forg.highwire.dtl.DTLVardef@1f03355org.highwire.dtl.DTLVardef@ba1fc7org.highwire.dtl.DTLVardef@17c5de2_HPS_FORMAT_FIGEXP M_FIG C_FIG The nucleoid-associated proteins Dps, H-NS, and HU shape the bacterial chromosome in the deep stationary phase through their interactions with the nucleoid.

Testicular germ cell tumours (TGCTs) are the most common cancer in young males and are considered curable if they respond to platinum-based therapy. However, a significant number of refractory patients develop metastatic disease and the lack targeted therapy remains an unmet clinical need. To identify novel therapeutic targets, we investigated the epigenetic instability of TGCTs and characterised novel oncogenic gene networks regulated by transposable elements (TEs)-derived long noncoding RNAs (lncRNAs) which are controlled by PIWI-interacting RNAs (piRNAs). A TGCT-specific piRNA signature was identified by bioinformatics analysis of the The Cancer Genome Atlas (TCGA) TGCT dataset and analysis of piRNAs mapped to active LINE1 sequences identified piR-hsa-7221 as a transcriptional regulator of the lncRNA CASC9 in seminoma tumours. We show that piR-hsa-7221 binds to a complementary LINE1 LIPA5 sequence and regulates the expression of CASC9 driven by the LINE1 antisense promoter. Therefore, loss of piR-hsa-7221 drives the upregulation and oncogenic activity of CASC9, which as is impaired after silencing, leading to reduced cancer cell proliferation and invasion, as well as increased sensitivity to cisplatin treatment. These effects are associated with the regulation of the cell cycle, developmental pathways, extracellular matrix, hormone metabolism and immune responses, highlighting WNT signalling as a significant downstream target. Therefore, this novel epigenetic mechanism provides new insights into the role of piRNA-mediated regulation of oncogenic lncRNAs derived from active transposable elements. Importantly, the identification of piR-hsa-7221 and the lncRNA CASC9, together with the associated gene networks highlights novel therapeutic targets for the treatment of seminoma TGCTs.

Essential nuclear processes require pairs of chromosomal loci to find each other in three-dimensional space. Polymer models of chromosome dynamics typically assume that the stochastic forces driving such locus motion are spatially uncorrelated, implying that relative diffusion follows directly from single-locus dynamics. Here we show that this assumption fails in living cells. Using live-cell imaging in fly embryos and mouse embryonic stem cells, we find that pairwise locus distances diffuse markedly slower than predicted for independent fluctuations. Combining stochastic trajectory analysis with polymer simulations, we demonstrate that this slowdown arises from non-equilibrium spatially correlated fluctuations (SCFs) in the nucleoplasm, which cause nearby loci to move coherently. We establish three experimentally testable signatures of SCFs: fluctuation amplitudes plateau at large distances, are independent of genomic separation, and show an anomalous temporal scaling. All three predictions are confirmed experimentally, including for loci on separate chromosomes. ATP depletion and disruption of cohesin-mediated loop extrusion reveal that both active processes and crosslinking contribute to correlation magnitudes. Because SCFs slow relative motion preferentially at short distances, they reduce encounter frequencies while prolonging encounter durations, generating a trade-off with direct implications for gene regulation. Our results identify spatially correlated fluctuations as a fundamental determinant of relative motion in confined active polymers.

Envelope-Limited Chromatin Sheets (ELCS) can be induced in human promyelocytic HL-60/S4 cells by treatment with retinoic acid (RA). After 4 days, the differentiated granulocytes exhibit multilobed nuclei with outgrowths of the nuclear envelope (NE) and associated heterochromatin extending into the surrounding cytoplasm (ELCS). These fascinating structures reveal a periodic meshwork of 30 nm chromatin fibers, when viewed by Cryo-electron microscopy. Genetic and biochemical evidence indicates that RA increases the synthesis of Lamin B Receptor (LBR), which is a key enzyme for Cholesterol biosynthesis and is an essential bridge between the NE and peripheral heterochromatin. This article is in part a review of our microscopic data on the structure of ELCS, and in part a description of related transcription changes that result in the formation of ELCS. In addition, this article contains a structural and biochemical comparison of RA-induced granulocytes with phorbol ester (TPA) induced HL-60/S4 macrophages, which lack nuclear lobulation, do not form ELCS, and exhibit a reduction in LBR and Cholesterol biosynthesis. From our perspective, ELCS can be viewed as "fabric" outgrowths of the nuclear envelope, frequently connecting nuclear lobes and capable of sustaining the twisting and squeezing distortions imposed upon nuclear shape, as the granulocytes traverse narrow tissue channels.

DNA-binding proteins must quickly locate specific sites on DNA to enable replication, repair, and transcription. While sequence-specific recognition is well understood, the physical basis of structure-specific recognition remains unclear, limiting our understanding of DNA damage repair. Proteins must distinguish damaged sites within largely undamaged DNA; however, studying this is challenging due to DNAs dynamic nature. We hypothesised that DNA damage causes changes in DNA structure, signalling protein recruitment. Using confocal single-molecule FRET, we analysed seven DNA duplexes containing modifications such as ribonucleotide, 8-oxoguanine (8-oxoG), abasic sites, nicks, and gaps, which are all involved in the base excision repair (BER) pathway. Each construct was measured with nine dye pairs in triplicate to capture changes in bending, twisting, and stretching. An automated analysis pipeline processed 162 measurements, enabling rigorous statistical comparisons. All modifications altered FRET efficiencies compared to undamaged DNA, including the subtlest change: a single oxygen difference (ribo-vs deoxyribonucleotide). Abasic sites, nicks, and gaps had the greatest effects. These findings provide direct evidence that DNA damage affects duplex structure and dynamics beyond the lesion site, suggesting DNA flexibility changes may act as an early signal for repair protein recruitment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/709887v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@a85839org.highwire.dtl.DTLVardef@3813dborg.highwire.dtl.DTLVardef@19fa06aorg.highwire.dtl.DTLVardef@dc9729_HPS_FORMAT_FIGEXP M_FIG C_FIG

Specific interchromosomal interactions indicate direct and nonrandom physical associations between pairs of genome positions on two different chromosomes. These contact interactions can be direct communication between non-homologous chromosomes and can enable coordinated activities. It is useful to annotate these complex contact interaction patterns and render them to a property associated with a single genome position, both for a clean visualization of the patterns and for facilitating the comparison with linear genomic annotations and underpinning biological functions. We utilize abstract graphs to characterize interchromosomal interaction, as network analysis may succinctly summarize complex interaction structures. We built a graph representation of cross-chromosomal contact interactions derived from Hi-C data and implemented three network-based annotations which consistently indicate the interchromosomal interaction strength associated with specific genomic positions. Equipped with these metrics, we further investigate whether a chromosome relies on shared hot spots to communicate with other chromosomes. We found that half of the strong interaction positions of chromosome 19 are shared for interacting with chromosomes 17 and 22. We further found that lamina-associated domains (LADs) participate in fewer interchromosomal contacts. Overall, the network-based annotation framework reveals distinct chromosome regulation patches and provides insight into how chromosomes associate with each other and organize with respect to the nuclear envelope.

In multi-cellular eukaryotic organisms, cell type and specific functional identity are defined by the epigenetic patterning of chemical modifications to DNA and chromatin that modulate the expression and silencing of specific genes. When a cell divides, histones containing important epigenetic marks are distributed between the two daughter strands leading to a temporary dilution of epigenetic information and cell identity. In this work we introduce a physics-based model of epigenetic memory that explains how cells restore and maintain H3K9me3 and H3K27me3 histone methylation patterning after cell division. We demonstrate that emergence and maintenance of the epigenetic program is driven by an evolved mechanism that makes use of the biophysics of polymers, phase condensates and enzymatic activity. We validate our model via genome-wide epigenetic time-course simulation and comparison to experimental epigenetic data from multiple donors, multiple cell types, and for multiple epigenetic marks. Finally, we use our model as a conceptual framework to understand cellular reprogramming by hypothesizing that these processes first contend with and later utilize somatic epigenetic maintenance programs.

Nucleus shape is a sensitive indicator of cell state, influenced by numerous bio-chemical and physiological factors. While prior work has cataloged how perturbations alter nucleus morphology, we address the inverse: inferring underlying molecular changes from nucleus shape alone. We previously developed a mechanical model yielding two nondimensional parameters: flatness index and scale factor, which are surrogate measures for cortical actin tension and nuclear envelope compliance respectively. In this study, we apply these parameters to investigate the dynamics in cellular mechanics during confined migration. We fabricated polydimethylsiloxane (PDMS) microchannels with widths of 3 {micro}m (high confinement) and 10 {micro}m (low confinement) and tracked cells migrating through them. We captured high-frequency 3D nucleus shapes via double fluorescence exclusion microscopy and custom image analysis. Fitting the model and estimating flatness index and scale factor to time-resolved shapes revealed dynamic regulation in 3 {micro}m channels: actin tension decreased and nucleus compliance increased immediately before nucleus entry into the constriction, with rapid restoration to baseline upon exit. No such changes occurred in 10 {micro}m channels, indicating active, confinement-dependent cytoskeletal adaptation. Immunostaining for YAP and lamin-A,C confirmed these model inferences. Our results uncover mechanostasis, active mechanical homeostasis, during confined migration and establish the combination of double fluorescence exclusion microscopy and nondimensional nucleus shape parameters as a powerful, non-invasive tool for single-cell mechanobiology studies.

Somatic homolog pairing is a defining feature of the diploid genome organization in Drosophila and underlies its transvection-based gene regulation. Here, to understand the physical effect of homolog pairing on the resulting three-dimensional (3D) organization, we employ the heterogeneous loop model and reconstruct 3D structures of the Drosophila embryo genome based on its haplotype-resolved Hi-C data. The resulting structures reveal robust end-to-end juxtaposition between homologous chromosomes amid substantial cell-to-cell variability. On sub-megabase scales, tight pairing between homologous loci at domain boundaries give rise to significant coincidence between cis and trans-homolog domain boundaries in the Hi-C map, while interior regions remain loosely associated. To uncover the physical origin of this organization, we compare the contact maps resulting from the polymer models implementing specific and non-specific button-mediated pairing mechanisms with Hi-C, finding that the intra-chromosomal contacts constrained by specifically paired inter-chromosomal buttons give rise to pairing-induced domains (PIDs). Our study suggests specific adhesive interactions as a central organizing principle of the diploid genome in Drosophila embryos. SignificanceSomatic homolog pairing distinguishes Drosophila from most eukaryotes; yet how the homolog pairing organizes Drosophila genome has remained elusive due to the lack of explicit model. By analyzing 3D structures reconstructed from haplotype-resolved Hi-C data, we clarify that specific homolog-recognizing buttons should generate pairing-induced domains that simultaneously organize cis and trans-homolog contacts. Our study provides a physical explanation on how a single molecular mechanism can simultaneously coordinate homolog pairing and architecture of chromatin domain in the diploid genome.

Genomes are organized across several hierarchical levels. Intracellular phase transitions compartmentalize many genomic processes, including transcription and DNA repair. However, little is known regarding how phase transitions contribute to replication, in which long strands of double- and single-stranded DNA need to be coordinated. Here, we investigated the molecular interactions driving the condensation of core mitochondrial replication components into mt-nucleoids. Complex phase behavior emerged among purified mt-replication components: each nucleic acid colocalized with its cognate architectural protein within multiphasic condensates. Using single-molecule experiments, we found that formation of ssDNA increased the partitioning of its cognate protein mtSSB within the condensate, consistent with the preferential localization of mtSSB to replicating mt-nucleoids. To develop mechanistic insights, we built a minimalistic coarse-grained model of mt-replication components that showed how interactions between binary pairs dictate their assembly within condensates. The multiphasic organization of mt-nucleoids has implications for how replication can be spontaneously organized in cells. HighlightsO_LIComplex co-phase behavior of TFAM-mtDNA depends on their affinity and DNA length C_LIO_LIMt-replication architectural proteins segregate ssDNA and dsDNA within condensates C_LIO_LImtSSB selectively partitions into actively replicating nucleoids in vivo C_LIO_LIExposure of ssDNA promotes mtSSB partitioning into TFAM-mtDNA condensates C_LI

The mitotic spindle is a biomechanical structure whose length must be precisely controlled to ensure faithful chromosome segregation. The treadmilling of microtubules towards centrosomes, termed poleward flux, is involved in spindle length control. However, poleward flux has been shown to be both inversely and directly proportional to spindle length, a contradiction that remains unexplained by current mechanisms. Here we introduce a model which demonstrates that length-dependent regulation at both microtubule ends allows plus- and minus-end dynamics to synchronize with one another, enabling pathways of poleward flux-based spindle length control which can rectify previous results. Moreover, our model predicts that spindle length and poleward flux can vary independently via simultaneous perturbations at both microtubule ends, which we experimentally validate with combinations of KIF18A, KIF2A, and KATNB1 depletions in human cells. Our results thus resolve a longstanding paradox and provide mechanistic insight into the reciprocal control of spindle length and poleward flux.

Variants in ACTB gene encoding for cytoplasmic {beta}-actin result in a group of rare disorders called non-muscle actinopathies (NMA). We investigated the cellular effects of a missense variant, G302A, and a four-amino-acid deletion, S338-I341, associated with the subgroup of NMA - ACTB pLoF (predicted loss-of-function) disorder in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the mechanically-induced reorganization of the actin cytoskeleton, we found that G302A mutant cells showed more dense actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of the deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was drastically reduced. We could partially rescue this fascinating effect by overexpressing the active form of the formin mDia. Our findings open the possibility to validate the cellular phenotype in the most affected patients cells, in neurons.