Biology subjects

Safran, S.

Publications and source records attributed to Safran, S..

8 recordsLinked to original sources

bioRxiv2026

Chromatin tethering to the nuclear envelope enhances its accessibility to RNAPII and promotes chromatin asymmetric organization

The intrinsic tendency of chromatin to self-attract competes with its association with RNA Polymerase II (RNAPII), a prerequisite for efficient transcription. Using high-resolution live imaging of chromatin and RNAPII organization in Drosophila larval muscle nuclei, we demonstrate that chromatin tethering to the nuclear envelope via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is essential for maintaining chromatin three-dimensional organization. Disruption of chromatin-lamina interactions either in LINC mutants or following knockdown of Barrier-to-Autointegration Factor (BAF) results in enhanced chromatin clustering in the nucleoplasm and reduced RNAPII-chromatin interaction. Consistent with these observations, computer simulations revealed an inverse relationship between the chromatin cluster size and the degree of chromatin tethering to the nuclear lamina. We also measured chromatin distribution with respect to the nuclear lamina and found asymmetry of RNAPII distributions within chromatin clusters, which correlated with their proximity to the nuclear envelope, a relationship that is lost in nuclei lacking a functional LINC complex. Our findings demonstrate that chromatin association with the nuclear envelope counteracts chromatin self-attraction and facilitates RNAPII binding to DNA.

molecular biology↗

bioRxiv2024

HP1a promotes chromatin liquidity and drives spontaneous heterochromatin compartmentalization

Compartmentalization of the nucleus into heterochromatin and euchromatin is highly conserved across eukaryotes. Constitutive heterochromatin (C-Het) constitutes a liquid-like condensate that packages the repetitive regions of the genome through the enrichment of histone modification H3K9me3 and recruitment of its cognate reader protein Heterochromatin Protein-1 (HP1a). The ability for well-ordered nucleosome arrays and HP1a to independently form biomolecular condensates suggests that the emergent material properties of C-Het compartments may contribute to its functions such as force-buffering, dosage-dependent gene silencing, and selective permeability. Using an in vitro reconstitution system we directly assess the contributions of H3K9me3 and HP1a on the biophysical properties of C-Het. In the presence of HP1a, H3K9me3 (Me-) and unmodified (U-) chromatin form co-condensates composed of distinct, immiscible domains. These chromatin domains form spontaneously and are reversible. Independently of HP1a, H3K9me3 modifications are sufficient to increase linker-DNA length within chromatin arrays and slow chromatin condensate growth. HP1a increases the liquidity of chromatin condensates while dramatically differentiating the viscoelastic properties of Me-chromatin versus U-chromatin. Mutating key residues in HP1a show that HP1a interactions with itself and chromatin determine the relative interfacial tension between chromatin compartments, however the formation of condensates is driven by the underlying chromatin. These direct measurements map the energetic landscape that determines C-Het compartmentalization, demonstrating that nuclear compartmentalization is a spontaneous and energetically favorable process in which HP1a plays a critical role in establishing a hierarchy of affinities between H3K9me3-chromatin and unmodified-chromatin. Highlights HP1a is necessary and sufficient for heterochromatin compartmentalization. Heterochromatin compartmentalization is reversible and proceeds through microphase-separation. H3K9me3 is sufficient to change nucleosome-array dynamics and mesoscale material properties. HP1a increases chromatin liquidity. HP1a-chromatin interaction modes tune the interfacial tensions and morphologies of heterochromatin compartments.

biophysics↗

bioRxiv2023

Affinity hierarchies underlie the co-assembly of nucleolar and heterochromatin condensates in Drosophila embryos

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. Nucleoli are the sites of ribosome synthesis, while the repeat-rich PCH is essential for chromosome segregation, genome stability, and transcriptional silencing. How and why these two distinct condensates co-assemble is unclear. Here, using high-resolution live imaging of Drosophila embryogenesis, we find that de novo establishment of PCH around the nucleolus is highly dynamic, transitioning from the nuclear edge to surrounding the nucleolus. Eliminating the nucleolus by removing the ribosomal RNA genes (rDNA) resulted in increased PCH compaction and subsequent reorganization into a toroidal structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or neocondensates, including enrichment in the PCH toroidal hole. Combining these observations with physical modeling revealed that nucleolar-PCH associations can be mediated by a hierarchy of interaction strengths between PCH, nucleoli, and amphiphilic protein(s) that have affinities for both nucleolar and PCH components. We validated this model by identifying a candidate amphiphile, a DEAD-Box RNA Helicase called Pitchoune, whose depletion or mutation of its PCH interaction motif disrupted PCH-nucleolar associations. Together, this study unveils a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrates that nucleoli are required for normal PCH organization, and identifies Pitchoune as an amphiphilic molecular link required for PCH-nucleolar associations.

cell biology↗

bioRxiv2022

Polymer dynamics relates chromosome mixing to temporal changes in biological contact maps

Chromosomes are arranged in distinct territories within the nucleus of animal cells. Recent experiments have shown that these territories overlap at their edges, suggesting partial mixing during interphase. Genomewide, biological contact maps in humans and Drosophila show only a low degree of contact between different chromosomes; however, the mixing in yeast is considerably higher. Recent theoretical estimates considered topological mixing of chromosomes by polymer reptation, and suggested that the time scale for chromosome mixing is years. This implies that a cell will typically divide before its chromosomes mix by reptation. Here, we use a generic polymer simulation to quantify the dynamics of chromosome mixing over time. We introduce the chromosome mixing index that quantifies the mixing of distinct chromosomes in the nucleus. We find that the chromosome mixing index increases as a power-law with time, and the scaling exponent varies non-monotonically with self-interaction and volume fraction. By comparing the chromosome mixing index with both subdiffusion due to (nontopological) intermingling of chromosomes as well as longer-time reptation, we show that the scaling exponent of the chromosome mixing index is related to intermingling for relatively small chromosome attractions and to reptation for large attractions. The model is extended to realistic biological conditions.

biophysics↗

bioRxiv2021

Balance of osmotic pressures determines the volume of the cell nucleus

The volume of the cell nucleus varies across cell-types and species, and is commonly thought to be determined by the size of the genome and degree of chromatin compaction. However, this notion has been challenged over the years by multiple experimental evidence. Here, we consider the physical condition of mechanical force balance as a determining condition of the nuclear volume and use quantitative, order-of-magnitude analysis to estimate the forces from different sources of nuclear and cellular pressure. Our estimates suggest that the dominant pressure within the nucleus and cytoplasm originates from the osmotic pressure of proteins and RNA molecules that are localized to the nucleus or cytoplasm by out-of-equilibrium, active nucleocytoplasmic transport rather than from chromatin or its associated ions. This motivates us to formulate a physical model for the ratio of the cell and nuclear volumes in which osmotic pressures of localized proteins determine the relative volumes. In accordance with unexplained observations that are century-old, our model predicts that the ratio of the cell and nuclear volumes is a constant, robust to a wide variety of biochemical and biophysical manipulations, and is changed only if gene expression or nucleocytoplasmic transport are modulated.

biophysics↗

bioRxiv2021

Physical theory of biological noise buffering by multi-component phase separation

Maintaining homeostasis is a fundamental characteristic of living systems. In cells, this is contributed to by assembly of biochemically-distinct organelles, many of whom are not membrane-bound, but form by the physical process of liquid-liquid phase separation (LLPS). By analogy with LLPS in binary solutions, cellular LLPS was hypothesized to contribute to homeostasis by facilitating "concentration buffering", which renders the local protein concentration within the organelle robust to global variations in the average, cellular concentration (e.g. due to expression noise). Interestingly, concentration buffering was experimentally measured in vivo, in a simple organelle with a single solute, while it was observed not to be obeyed in one with several solutes. Here, we formulate theoretically and solve analytically a physical model of LLPS in a ternary solution of two solutes (A and B) that interact both homotypically (A-A attractions) and heterotypically (A-B attractions). Our physical theory predicts how the equilibrium concentrations in LLPS are related to expression noise and thus generalizes the concept of concentration buffering to multi-component systems. This allows us to reconcile the seemingly contradictory experimental observations. Furthermore, we predict that incremental changes of the homotypic and heterotypic interactions among the molecules that undergo LLPS, such as those that are caused by mutations in the genes encoding the proteins, may increase the efficiency of concentration buffering of a given system. Thus, we hypothesize that evolution may optimize concentration buffering as an efficient mechanism to maintain LLPS homeostasis, and suggest experimental approaches to test this in different systems. SignificanceThe stochastic nature of transcription/translation implies that the concentrations of cellular proteins are "noisy" and not constant in time or across cell populations. Liquid-liquid phase separation (LLPS), can reduce or "buffer" this noise by maintaining well-defined concentrations, even in the presence of concentration distributions. However, this idea was recently challenged experimentally in multicomponent systems. Our physical theory of LLPS in ternary systems (solutes A and B in a solvent), predicts their LLPS properties as a function of the A-A (homotypic) and A-B (heterotypic) interaction strengths. We show how buffering can be effective if the noise distribution aligns with the tie-lines of the phase diagram and suggest that evolution may optimize concentration buffering by selecting mutations that lead to this.

biophysics↗

bioRxiv2020

Mesoscale phase separation of chromatin in the nucleus

Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the compositional profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.

biophysics↗

bioRxiv2020

Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

Packaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin self-attractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior. This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

cell biology↗