RESUMEN
Coarsening is a ubiquitous phenomenon in droplet systems near thermodynamic equilibrium-as an increase in droplet size lowers the system's free energy-however, coarsening of droplets in nonequilibrium systems, such as the cell nucleus, is far from understood. Liquid condensates in the cell nucleus, like nucleoli, form by liquid-liquid phase separation and play a key role in the nuclear organization. In human cells, nucleolar droplets are nucleated at the beginning of the cell cycle and coarsen with time by coalescing with each other. Upon coarsening, human nucleoli exhibit an anomalous volume distribution P(V)â¼V-1, which cannot be explained by any existing theory. In this work, we investigate physical mechanisms behind the anomalous coarsening of human nucleoli. Using spinning disk confocal microscopy, we simultaneously record dynamic behavior of nucleoli and their surrounding chromatin before their coalescence in live human cells. We find that nucleolar anomalous coarsening persists during the entire cell cycle. We measure chromatin flows and density between and around nucleoli, as well as relative motion of two nucleoli before they coalesce. We find that, before nucleolar coalescence, chromatin concentration decreases in the space between nucleoli and the nucleoli move faster toward each other, resembling an effective depletion attraction between the coalescing nucleoli. Indeed, our computational simulations of nucleolar dynamics show that short-ranged attraction is sufficient to explain the observed anomalous volume distribution of human nucleoli. Overall, our results reveal a potential physical mechanism contributing to coarsening of human nucleoli. Such knowledge expands our picture of the physical behavior of liquid condensates inside the cell nucleus and our understanding of the dynamic nuclear organization.
Asunto(s)
Nucléolo Celular , Cromatina , Humanos , Nucléolo Celular/metabolismo , Cromatina/metabolismo , Ciclo Celular , Células HeLaRESUMEN
Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124 cis-target genes of 91 noncoding blood trait GWAS loci. Using precise variant insertion through base editing, we connected specific variants with gene expression changes. We also identified trans-effect networks of noncoding loci when cis target genes encoded transcription factors or microRNAs. Networks were themselves enriched for GWAS variants and demonstrated polygenic contributions to complex traits. This platform enables massively parallel characterization of the target genes and mechanisms of human noncoding variants in both cis and trans.
Asunto(s)
Enfermedad , Estudio de Asociación del Genoma Completo , Herencia Multifactorial , Sitios de Carácter Cuantitativo , Análisis de la Célula Individual , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Proteómica , Células Sanguíneas , RNA-Seq , Enfermedad/genéticaRESUMEN
Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias , Biología , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica , Genoma , Humanos , Neoplasias/genética , Neoplasias/terapiaRESUMEN
Material properties of the genome are critical for proper cellular function - they directly affect timescales and length scales of DNA transactions such as transcription, replication and DNA repair, which in turn impact all cellular processes via the central dogma of molecular biology. Hence, elucidating the genome's rheology in vivo may help reveal physical principles underlying the genome's organization and function. Here, we present a novel noninvasive approach to study the genome's rheology and its response to mechanical stress in form of nuclear injection in live human cells. Specifically, we use Displacement Correlation Spectroscopy to map nucleus-wide genomic motions pre/post injection, during which we deposit rheological probes inside the cell nucleus. While the genomic motions inform on the bulk rheology of the genome pre/post injection, the probe's motion informs on the local rheology of its surroundings. Our results reveal that mechanical stress of injection leads to local as well as nucleus-wide changes in the genome's compaction, dynamics and rheology. We find that the genome pre-injection exhibits subdiffusive motions, which are coherent over several micrometers. In contrast, genomic motions post-injection become faster and uncorrelated, moreover, the genome becomes less compact and more viscous across the entire nucleus. In addition, we use the injected particles as rheological probes and find the genome to condense locally around them, mounting a local elastic response. Taken together, our results show that mechanical stress alters both dynamics and material properties of the genome. These changes are consistent with those observed upon DNA damage, suggesting that the genome experiences similar effects during the injection process.
Asunto(s)
ADN , Genoma Humano , Núcleo Celular , ADN/genética , Humanos , Reología , Estrés MecánicoRESUMEN
Liquid-liquid phase separation (LLPS) has been recognized as one of the key cellular organizing principles and was shown to be responsible for formation of membrane-less organelles such as nucleoli. Although nucleoli were found to behave like liquid droplets, many ramifications of LLPS including nucleolar dynamics and interactions with the surrounding liquid remain to be revealed. Here, we study the motion of human nucleoli in vivo, while monitoring the shape of the nucleolus-nucleoplasm interface. We reveal two types of nucleolar pair dynamics: an unexpected correlated motion prior to coalescence and an independent motion otherwise. This surprising kinetics leads to a nucleolar volume distribution, [Formula: see text], unaccounted for by any current theory. Moreover, we find that nucleolus-nucleoplasm interface is maintained by ATP-dependent processes and susceptible to changes in chromatin transcription and packing. Our results extend and enrich the LLPS framework by showing the impact of the surrounding nucleoplasm on nucleoli in living cells.
Asunto(s)
Nucléolo Celular/química , Núcleo Celular/química , Cromatina/genética , Proteínas Nucleares/química , Adenosina Trifosfato/química , Nucléolo Celular/genética , Núcleo Celular/genética , Cromatina/química , Humanos , Cinética , Proteínas Nucleares/genéticaRESUMEN
The nucleolus is a membraneless organelle embedded in chromatin solution inside the cell nucleus. By analyzing surface dynamics and fusion kinetics of human nucleoli in vivo, we find that the nucleolar surface exhibits subtle, but measurable, shape fluctuations and that the radius of the neck connecting two fusing nucleoli grows in time as r(t)â¼t^{1/2}. This is consistent with liquid droplets with low surface tension â¼10^{-6} N m^{-1} coalescing within an outside fluid of high viscosity â¼10^{3} Pa s. Our study presents a noninvasive approach of using natural probes and their dynamics to investigate material properties of the cell and its constituents.
Asunto(s)
Nucléolo Celular/fisiología , Núcleo Celular/fisiología , Modelos Biológicos , Nucléolo Celular/química , Núcleo Celular/química , Cromatina/química , Cromatina/fisiología , Células HeLa , Humanos , ReologíaRESUMEN
We model a spatially detailed, two-sex population dynamics, to study the cost of ecological restoration. We assume that cost is proportional to the number of individuals introduced into a large habitat. We treat dispersal as homogeneous diffusion in a one-dimensional reaction-diffusion system. The local population dynamics depends on sex ratio at birth, and allows mortality rates to differ between sexes. Furthermore, local density dependence induces a strong Allee effect, implying that the initial population must be sufficiently large to avert rapid extinction. We address three different initial spatial distributions for the introduced individuals; for each we minimize the associated cost, constrained by the requirement that the species must be restored throughout the habitat. First, we consider spatially inhomogeneous, unstable stationary solutions of the model's equations as plausible candidates for small restoration cost. Second, we use numerical simulations to find the smallest rectangular cluster, enclosing a spatially homogeneous population density, that minimizes the cost of assured restoration. Finally, by employing simulated annealing, we minimize restoration cost among all possible initial spatial distributions of females and males. For biased sex ratios, or for a significant between-sex difference in mortality, we find that sex-specific spatial distributions minimize the cost. But as long as the sex ratio maximizes the local equilibrium density for given mortality rates, a common homogeneous distribution for both sexes that spans a critical distance yields a similarly low cost.