Transient Interphase Microtubules Appear in Differentiating Sponge Cells.

Microtubules are an indispensable component of all eukaryotic cells due to their role in mitotic spindle formation, yet their organization and number can vary greatly in the interphase. The last common ancestor of all eukaryotes already had microtubules and microtubule motor proteins moving along them. Sponges are traditionally regarded as the oldest animal phylum. Their body does not have a clear differentiation into tissues, but it contains several distinguishable cell types. The choanocytes stand out among them and are responsible for creating a flow of water with their flagella and increasing the filtering and feeding efficiency of the sponge. Choanocyte flagella contain microtubules, but thus far, observing a developed system of cytoplasmic microtubules in non-flagellated interphase sponge cells has been mostly unsuccessful. In this work, we combine transcriptomic analysis, immunofluorescence, and electron microscopy with time-lapse recording to demonstrate that microtubules appear in the cytoplasm of sponge cells only when transdifferentiation processes are activated. We conclude that dynamic cytoplasmic microtubules in the cells of sponges are not a persistent but rather a transient structure, associated with cellular plasticity.

Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization.

In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets.

The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Activity-driven chromatin organization during interphase: Compaction, segregation, and entanglement suppression.

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.

An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.

Transcription-induced active forces suppress chromatin motion.

The organization of interphase chromosomes in a number of species is starting to emerge thanks to advances in a variety of experimental techniques. However, much less is known about the dynamics, especially in the functional states of chromatin. Some experiments have shown that the motility of individual loci in human interphase chromosome decreases during transcription and increases upon inhibiting transcription. This is a counterintuitive finding because it is thought that the active mechanical force (F) on the order of ten piconewtons, generated by RNA polymerase II (RNAPII) that is presumably transmitted to the gene-rich region of the chromatin, would render it more open, thus enhancing the mobility. We developed a minimal active copolymer model for interphase chromosomes to investigate how F affects the dynamical properties of chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of F and are enhanced at small F values, which has also been observed in experiments. In the intermediate F, the bond length between consecutive loci increases, becoming commensurate with the distance at the minimum of the attractive interaction between nonbonded loci. This results in a transient disorder-to-order transition, leading to a decreased mobility during transcription. Strikingly, the F-dependent change in the locus dynamics preserves the organization of the chromosome at [Formula: see text]. Transient ordering of the loci, which is not found in the polymers with random epigenetic profiles, in the gene-rich region might be a plausible mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin.

Lamin A/C and PI(4,5)P2-A Novel Complex in the Cell Nucleus.

Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.

Structural basis for the preservation of a subset of topologically associating domains in interphase chromosomes upon cohesin depletion.

Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are present even without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer-promoter/promoter-promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

Culturing and Differentiation of Patient-Derived Ectocervical Epithelial Stem Cells Using Air-Liquid Interphase and Matrigel Scaffold.

The ectocervix acts as a multilayered defense barrier, protecting the female reproductive system from external pathogens and supporting fertility and pregnancy. To understand the complex cellular and molecular mechanisms of cervical biology and disease, reliable in vitro models are vital. We present an efficient method to isolate and cultivate epithelial stem cells from ectocervical tissue biopsies. This method combines enzymatic digestion, mechanical dissociation, and selective culturing to obtain pure ectocervical epithelial cells for further investigation. The protocol accommodates both 2D stem cell monolayer and advanced 3D culture systems, such as air-liquid interface and Matrigel scaffolds, using a defined media cocktail, making it highly versatile. The primary ectocervical epithelial cells retain their native characteristics, enabling the exploration of ectocervical epithelial tissue behavior and pathology. This chapter provides step-by-step guidelines for setting up 2D and 3D cultures, facilitating adoption across different laboratories, and advancing cervical biology and disease research.

Physical forces modulate interphase nuclear size.

The eukaryotic nucleus exhibits remarkable plasticity in size, adjusting dynamically to changes in cellular conditions such as during development and differentiation, and across species. Traditionally, the supply of structural constituents to the nuclear envelope has been proposed as the principal determinant of nuclear size. However, recent experimental and theoretical analyses have provided an alternative perspective, which emphasizes the crucial role of physical forces such as osmotic pressure and chromatin repulsion forces in regulating nuclear size. These forces can be modulated by the molecular profiles that traverse the nuclear envelope and assemble in the macromolecular complex. This leads to a new paradigm wherein multiple nuclear macromolecules that are not limited to only the structural constituents of the nuclear envelope, are involved in the control of nuclear size and related functions.

Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

Fluorescence in situ hybridisation for interphase chromosomal aberration-based biological dosimetry.

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8 Gy of gamma radiation. Irradiated whole blood was kept for 2 h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8 Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8 Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.

The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.

Ultrastructure and fractal property of chromosomes in close-to-native yeast nuclei visualized using X-ray laser diffraction.

Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300 nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800 nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400-20 nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58 nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1 mm in 800 nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

Analysis of human invasive cytotrophoblasts demonstrates mosaic aneuploidy.

A total of 24 chromosome-specific fluorescence in situ hybridization probes for interphase nucleus analysis were developed to determine the chromosomal content of individual human invasive cytotrophoblasts derived from in vitro cultured assays. At least 75% of invasive cytotrophoblasts were hyperdiploid and the total number of chromosomes ranged from 47 to 61. The results also demonstrated that these hyperdiploid invasive cytotrophoblasts showed significant heterogeneity. The most copy number gains were observed for chromosomes 13, 14, 15, 19, 21, and 22 with average copy number greater than 2.3. A parallel study using primary invasive cytotrophoblasts also showed a similar trend of copy number changes. Conclusively, 24-chromosome analysis of human non-proliferating cytotrophoblasts (interphase nuclei) was achieved. Hyperdiploidy and chromosomal heterogeneity without endoduplication in invasive cytotrophoblasts may suggest a selective advantage for invasion and short lifespan during normal placental development.

Dynamic gel as artificial interphase layer for ultrahigh-rate and large-capacity lithium metal anode.

Constructing a stable artificial solid-electrolyte interphase has become one of the most effective strategies to overcome the poor reversibility of lithium metal anode, yet the protection role is still insufficient at elevated current densities over 10 mA cm-2 and large areal capacities over 10 mAh cm-2. Herein, we propose a dynamic gel with reversible imine groups, which is prepared via a cross linking reaction between flexible dibenzaldehyde-terminated telechelic poly(ethylene glycol) and rigid chitosan, to fabricate a protective layer for Li metal anode. The as-prepared artificial film shows combined merits of high Young's modulus, strong ductility and high ionic conductivity. When the artificial film is fabricated on a lithium metal anode, the thin protective layer shows a dense and uniform surface owing to the interactions between the abundant polar groups and lithium metal. Besides, the polar groups in the artificial film can homogenize the distribution of Li+ at the electrode/electrolyte interface. As a result, cycle stability over 3200 h under an areal capacity of 10 mAh cm-2 and a current density of 10 mA cm-2 has been obtained for the protected lithium metal anodes. Moreover, cycling stability and rate capability has been also improved in the full cells.

The influence of asymmetrical bipolar pulses and interphase intervals on the bipolar cancellation phenomenon in the ovarian cancer cell line.

The application of negative polarity electrical pulse (↓) following positive polarity pulses (↑) may induce bipolar cancellation (BPC), a unique physiological response believed to be specific to nanosecond electroporation (nsEP). The literature lacks analysis of bipolar electroporation (BP EP) involving asymmetrical sequences composed of nanosecond and microsecond pulses. Moreover, the impact of interphase interval on BPC caused by such asymmetrical pulse needs consideration. In this study, the authors utilized the ovarian clear carcinoma cell line (OvBH-1) model to investigate the BPC with asymmetrical sequences. Cells were exposed to pulses delivered in 10-pulse bursts but as uni- or bipolar, symmetrical, or asymmetrical sequences with a duration of 600 ns or 10 µs and electric field strength equal to 7.0 or 1.8 kV/cm, respectively. It was shown that the asymmetry of pulses influences BPC. The obtained results have also been investigated in the context of calcium electrochemotherapy. The reduction of cell membrane poration, and cell survival have been observed following Ca2+ electrochemotherapy. The effects of interphase delays (1 and 10 µs) on the BPC phenomenon were reported. Our findings show that the BPC phenomenon can be controlled using pulse asymmetry or delay between the positive and negative polarity of the pulse.