Biology subjects

Rosen, M. K.

Publications and source records attributed to Rosen, M. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Spindle pole proteins confine chromosomes to ensure their expulsion during female meiosis

Animal oocytes undergo highly asymmetric divisions to expel excess copies of their genome into compact cells called polar bodies. This requires tight clustering and cortical positioning of meiotic chromosomes, yet the mechanism remains incompletely understood. Using C. elegans oocytes, we found that the meiotic spindle pole protein ZYG-9/ch-TOG delocalizes from microtubules to spread along the surface of chromosomes and prevent their dispersal in meiotic anaphase. This effect was more pronounced in the absence of the spindle, where ZYG-9 formed into a micron-scale droplet that enveloped all chromosomes. Purified ZYG-9 was sufficient to bind DNA and coat reconstituted chromatin. Mutations that perturb ZYG-9-DNA binding impaired chromosome packaging into polar bodies, resulting in oocytes carrying extra chromosomes and reduced fertility. We propose that liquid-like assemblies of spindle pole proteins are repurposed as surface-acting glue to tightly package meiotic chromosomes into polar bodies, thus ensuring oocytes have the correct genome copy number.

cell biology↗

bioRxiv2026

Electrostatic control of chromatin compaction safeguards against apoptotic DNA release

Apoptosis involves extensive intracellular reorganisation to facilitate the clearance of dying cells. A key step in this process is the destruction of the genome through fragmentation by caspase-activated endonuclease (CAD). Rather than dispersing after CAD-mediated cleavage, DNA fragments are compacted into a dense chromatin compartment. However, the underlying mechanism and biological relevance of this compaction remain unknown. Here we show that global deacetylation of histone tails promotes chromatin compaction during apoptosis, preventing DNA release into apoptotic extracellular vesicles. Using synthetic effectors that modulate nucleosome electrostatics independently of histone modifications, we demonstrate that electrostatic attraction alone is sufficient to compact and sequester fragmented chromatin. These findings reveal a mechanism by which global reprogramming of histone modifications coordinates fragmentation of the genome with its physical sequestration during apoptosis. Furthermore, our synthetic approach provides a tool to probe the role of physical forces in genome organisation across diverse biological contexts.

cell biology↗

bioRxiv2025

Near-atomistic simulations reveal the molecular principles that control chromatin structure and phase separation

Understanding how chromatins physicochemical properties shape its emergent organisation is central to deciphering genome function. To address this, we present OpenCGChromatin, a high-performance coarse-grained model that achieves near-atomistic simulations of chromatin systems an order of magnitude larger than previously possible, spanning biomolecular condensates and fibers tens of kilobases in length. OpenCGChromatin simulations independently predict, from physicochemical principles, the linker-DNA-dependent chromatin structures observed by cryo-ET and the relative thermodynamic stability of condensates inferred from biochemical assays. Crucially, OpenCGChromatin resolves histone-tail dynamics and interaction networks that remain inaccessible experimentally, explaining how linker-DNA length controls histone tail accessibility and the resulting multiscale structure of chromatin condensates. Extending simulations to 108-nucleosome fibers shows that acetylation disrupts chromatin compaction in a pattern-specific manner by weakening key tail-mediated interactions, with H4K16 and H3K9 emerging as the most energetically disruptive modifications. These results position OpenCGChromatin as a powerful framework for linking molecular detail to emergent chromatin organization.

biophysics↗

bioRxiv2025

An electrostatic repulsion model of centromere organisation

During cell division, chromosomes reorganise into compact bodies in which centromeres localise precisely at the chromatin surface1-4 to enable kinetochore-microtubule interactions essential for genome segregation5-8. The physical principles guiding this centromere positioning remain unknown. Here, we reveal that human core centromeres are directed to the chromatin surface by repulsion of centromere-associated proteins - independent of condensin-mediated loop extrusion and microtubule engagement. Using cellular perturbations, biochemical reconstitution, and multiscale molecular dynamics simulations, we show that chromatin surface localisation emerges from repulsion between condensed chromatin and both the kinetochore and the highly negatively charged centromere protein, CENP-B. Together, these elements form a centromeric region composed of two domains with opposing affinities, one favouring integration within the mitotic chromosome and the other favouring exposure to the surrounding cytoplasm, thereby driving surface positioning. Tethering synthetic negatively charged proteins to chromatin was sufficient to recapitulate this surface localisation in cells and in vitro, indicating that electrostatic repulsion is a key determinant of surface localisation. These findings demonstrate that centromere layering is not hardwired by chromatin folding patterns but instead emerges from phase separation in chromatin. Our work uncovers electrostatic polarity as a general and programmable mechanism to spatially organise chromatin.

cell biology↗

bioRxiv2025

Multi-scale structure of chromatin condensates rationalizes phase separation and material properties

Biomolecular condensates, compartments that concentrate molecules without surrounding membranes, are integral to numerous cellular processes. The structure and interaction networks of molecules within condensates remain poorly understood. Using cryo-electron tomography and molecular dynamics simulations we elucidated the structure of phase separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules both within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which in turn governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose non-random organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules ultimately determines physical properties of chromatin condensates, with implications for cellular chromatin organization.

biophysics↗

bioRxiv2024

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

biophysics↗

bioRxiv2024

Quantitative Spatial Analysis of Chromatin Biomolecular Condensates using Cryo-Electron Tomography

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation and function remain incompletely understood due to difficulties in visualizing the condensate interior at high resolution. Here we analyzed the structure of biochemically reconstituted chromatin condensates through cryo-electron tomography. We found that traditional blotting methods of sample preparation were inadequate, and high-pressure freezing plus focused ion beam milling was essential to maintain condensate integrity. To identify densely packed molecules within the condensate, we integrated deep learning-based segmentation with novel context-aware template matching. Our approaches were developed on chromatin condensates, and were also effective on condensed regions of in situ native chromatin. Using these methods, we determined the average structure of nucleosomes to 6.1 and 12 [A] resolution in reconstituted and native systems, respectively, and found that nucleosomes form heterogeneous interaction networks in both cases. Our methods should be applicable to diverse biochemically reconstituted biomolecular condensates and to some condensates in cells.

biophysics↗

bioRxiv2024

ExoSloNano: Multi-Modal Nanogold Tags for identi-fication of Macromolecules in Live Cells & Cryo-Electron Tomograms

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

biophysics↗

bioRxiv2024

Phospho-regulated tuning of viscoelastic properties balances centrosome growth and strength

Centrosomes are membranelles organelles containing centrioles encapsulated by pericentriolar material (PCM). PCM nucleates microtubules that help position and segregate chromosomes during mitosis, yet how PCM resists microtubule-mediated forces is poorly understood at the material level. Here, we show that PLK-1 phosphorylation of SPD-5 tunes the dynamics and material properties of the PCM scaffold in C. elegans embryos. Microrheology of reconstituted PCM condensates reveals that PLK-1 phosphorylation decreases SPD-5 dynamics and increases condensate viscoelasticity. Similarly, in embryos, phospho-mimetic SPD-5 is less dynamic than wild-type SPD-5, which itself is less dynamic than phospho-null SPD-5. PCM built with phospho-null SPD-5 is smaller than normal, but its assembly can be partially rescued by reducing microtubule-dependent forces. The same is true for PCM built with phospho-mimetic SPD-5, yet the underlying causes are distinct: under force, phospho-null SPD-5 fails to assemble, while phospho-mimetic SPD-5 forms hyper-stable foci that fail to cohere into a uniform, spherical mass. Both mutants have defects with chromosome segregation and viability. Thus, tuning of SPD-5 phosphorylation optimizes PCM material properties to achieve correct PCM size, integrity, and function. Our results demonstrate how regulated chemical modification of a scaffolding protein modulates the material properties and function of a membraneless organelle.

cell biology↗

bioRxiv2024

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, generically applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multi-point attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time FRAP imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates. Significance StatementThe understanding of biomolecular condensates has significantly benefited from biochemical reconstitution with microscopy detection. Here, we present a novel surface passivation method utilizing self-assembly of Pluronic F127 on hydrophobic surfaces. This approach not only effectively minimizes non-specific binding without altering the physical properties of the condensates but also offers universal passivation across a variety of condensate systems. It demonstrates high resistance to different treatments and enables condensate immobilization through controlled anchor points. This allows for highly sensitive analytical techniques, including single-molecule imaging. The simplicity and high-performance of this method, coupled with time and cost efficiencies, could facilitate robustness and throughput of experiments, and could broaden the accessibility of biochemical phase separation studies to a wider scientific community.

biochemistry↗

bioRxiv2023

Molecular features driving condensate formation and gene expression by the BRD4-NUT fusion oncoprotein are overlapping but distinct

Aberrant formation of biomolecular condensates has been proposed to play a role in several cancers. The oncogenic fusion protein BRD4-NUT forms condensates and drives changes in gene expression in Nut Carcinoma (NC). Here we sought to understand the molecular elements of BRD4-NUT and its associated histone acetyltransferase (HAT), p300, that promote these activities. We determined that a minimal fragment of NUT (MIN) in fusion with BRD4 is necessary and sufficient to bind p300 and form condensates. Furthermore, a BRD4-p300 fusion protein also forms condensates and drives gene expression similarly to BRD4-NUT(MIN), suggesting the p300 fusion may mimic certain features of BRD4-NUT. The intrinsically disordered regions, transcription factor-binding domains, and HAT activity of p300 all collectively contribute to condensate formation by BRD4-p300, suggesting that these elements might contribute to condensate formation by BRD4-NUT. Conversely, only the HAT activity of BRD4-p300 appears necessary to mimic the transcriptional profile of cells expressing BRD4-NUT. Our results suggest a model for condensate formation by the BRD4-NUT:p300 complex involving a combination of positive feedback and phase separation, and show that multiple overlapping, yet distinct, regions of p300 contribute to condensate formation and transcriptional regulation.

cell biology↗

bioRxiv2022

Small Molecule Properties Define Partitioning into Biomolecular Condensates

Biomolecular condensates regulate cellular function by compartmentalizing molecules without a surrounding membrane1,2. Condensate functions are believed to arise from the specific exclusion or enrichment of molecules3-5. Thus, understanding the principles governing condensate composition is critical to characterizing condensate function. The molecular bases of macromolecular composition have been studied in detail for several condensates6-9, but partitioning of small molecules into condensates remains poorly understood. Using mass spectrometry with validation by fluorescence microscopy, we quantified partitioning of [~]1700 metabolites and FDA-approved drugs into four condensates composed of different DNA and/or protein scaffolds. We found that partitioning spanned from [~]100-fold exclusion to [~]10,000-fold enrichment. Strong correlations between the different condensates suggest an underlying physical similarity despite disparate macromolecular components. Strongly partitioning molecules generally did not bind condensate-forming proteins with high affinity under conditions where condensates do not form, suggesting only a minor role for stereospecific interactions in partitioning. We developed a machine learning model that accurately predicts partitioning using only physicochemical properties of the compounds. The strongest predictors of partitioning were features related to aqueous solubility and hydrophobicity. Small molecule partitioning was similar for condensates in aqueous buffer and in concentrated cell extracts, suggesting analogous behaviors in vivo. Together, the data and model suggest that small molecule partitioning is not generally based on high-affinity, stereospecific interactions with scaffolds, but rather on physical properties of the compounds, and their compatibility with an emergent hydrophobic environment within the condensate. Our results will aid design of chemical compounds that target biomolecular condensates and reveal unexpected physical and functional similarities of distinct condensates.

biochemistry↗

bioRxiv2022

Quantitative reconstitution of yeast RNA processing bodies

Many biomolecular condensates appear to form through liquid-liquid phase separation (LLPS). Individual condensate components can often undergo LLPS in vitro, capturing some features of the native structures. However, natural condensates contain dozens of components with different concentrations, dynamics, and contributions to compartment formation. Most biochemical reconstitutions of condensates have not benefitted from quantitative knowledge of these cellular features nor attempted to capture natural complexity. Here, we build on prior quantitative cellular studies to reconstitute yeast RNA processing bodies (P bodies) from purified components. Individually, five of the seven highly-concentrated P-body proteins form homotypic condensates at cellular protein and salt concentrations, using both structured domains and intrinsically disordered regions. Combining the seven proteins together at their cellular concentrations with RNA, yields phase separated droplets with partition coefficients and dynamics of most proteins in reasonable agreement with cellular values. The dynamics of most proteins in the reconstitution are also comparable to cellular values. RNA delays the maturation of proteins within, and promotes reversibility of, P bodies. Our ability to quantitatively recapitulate the composition and dynamics of a condensate from its most concentrated components suggests that simple interactions between these components carry much of the information that defines the physical properties of the cellular structure.

biophysics↗

bioRxiv2022

Coupling of protein condensates to ordered lipid domains determines functional membrane organization

During T-cell activation, the transmembrane adaptor Linker of Activation of T-cells (LAT) forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. LAT has also been associated with cholesterol-rich condensed lipid domains. However, the potential coupling between protein condensation and lipid phase separation and its role in organizing T-cell signaling were unknown. Here, we report that LAT/Grb2/Sos1 condensates reconstituted on model membranes can induce and template lipid domains, indicating strong coupling between lipid- and protein-based phase separation. Correspondingly, activation of T-cells induces protein condensates that associate with and stabilize raft-like membrane domains. Inversely, lipid domains nucleate and stabilize LAT protein condensates in both reconstituted and living systems. This coupling of lipid and protein assembly is functionally important, since uncoupling of lipid domains from cytoplasmic protein condensates abrogates T-cell activation. Thus, thermodynamic coupling between protein condensates and ordered lipid domains regulates the functional organization of living membranes. SUMMARYMembrane-associated protein condensates couple to ordered membrane domains to determine the functional organization of T-cell plasma membranes

cell biology↗

bioRxiv2021

In Diverse Conditions Intrinsic Chromatin Condensates Have Liquid-like Material Properties

Eukaryotic nuclear DNA is wrapped around histone proteins to form nucleosomes, which further assemble to package and regulate the genome. Understanding of the physical mechanisms that contribute to higher order chromatin organization is limited. Previously, we reported the intrinsic capacity of chromatin to undergo phase separation and form dynamic liquid-like condensates, which can be regulated by cellular factors. Recent work from Hansen, Hendzel, and colleagues suggested these intrinsic chromatin condensates are solid in all but a specific set of conditions. Here we show that intrinsic chromatin condensates are fluid in diverse solutions, without need for specific buffering components. Exploring experimental differences in sample preparation and imaging between these two studies, we suggest what may have led Hansen, Hendzel, and colleagues to mischaracterize the innate properties of chromatin condensates. We also describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained movement of chromatin in cells.

biophysics↗

bioRxiv2021

Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails dramatic epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC.

cancer biology↗

bioRxiv2021

Synergistic Phase Separation of Two Pathways Promotes Integrin Clustering and Integrin Adhesion Complex Formation

Integrin adhesion complexes (IACs) are integrin-based plasma membrane-associated compartments where cells sense environmental cues. The physical mechanisms and molecular interactions that mediate nascent IAC formation are unclear. We found that both p130Cas ("Cas") and Focal adhesion kinase ("FAK") undergo liquid-liquid phase separation in vitro under physiologic conditions. Cas- and FAK- driven phase separation is sufficient to reconstitute kindlin-dependent integrin clustering in vitro. In vitro condensates and cellular IACs exhibit similar sensitivities to environmental perturbations including changes in temperature and pH. Furthermore, mutations that inhibit or enhance phase separation in vitro reduce or increase the number of IACs in cells, respectively. Finally, we find that the Cas and FAK pathways act synergistically to promote phase separation, integrin clustering and IAC formation in vitro and in cells. We propose that Cas- and FAK- driven phase separation provides an intracellular trigger for integrin clustering and nascent IAC formation.

cell biology↗

bioRxiv2021

A chromatin phase transition protects mitotic chromosomes against microtubule perforation

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. While condensin confers mechanical resistance towards spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and segregation mechanics of mitotic chromosomes. Here, we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with specific characteristics necessary for their precise movement during cellular division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. Hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules, and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin-intrinsic phase separation to genome segregation in dividing cells.

cell biology↗