High-content imaging-based pooled CRISPR screens in mammalian cells.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software µManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

DNA content contributes to nuclear size control in Xenopus laevis.

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.

Unravelling nuclear size control.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

The Chromatin-Associated Protein PWO1 Interacts with Plant Nuclear Lamin-like Components to Regulate Nuclear Size.

Spatial organization of chromatin contributes to gene regulation of many cellular processes and includes a connection of chromatin with the nuclear lamina (NL). The NL is a protein mesh that resides underneath the inner nuclear membrane and consists of lamins and lamina-associated proteins. Chromatin regions associated with lamins in animals are characterized mostly by constitutive heterochromatin, but association with facultative heterochromatin mediated by Polycomb-group (PcG) proteins has been reported as well. In contrast with animals, plant NL components are largely not conserved and NL association with chromatin is poorly explored. Here, we present the connection between the lamin-like protein, CROWDED NUCLEI1 (CRWN1), and the chromatin- and PcG-associated component, PROLINE-TRYPTOPHANE-TRYPTOPHANE-PROLINE INTERACTOR OF POLYCOMBS1, in Arabidopsis (Arabidopsis thaliana). We show that PWO1 and CRWN1 proteins associate physically with each other, act in the same pathway to maintain nuclear morphology, and control expression of a similar set of target genes. Moreover, we demonstrate that transiently expressed PWO1 proteins form foci located partially at the subnuclear periphery. Ultimately, as CRWN1 and PWO1 are plant-specific, our results argue that plants might have developed an equivalent, rather than homologous, mechanism of linking chromatin repression and NL.

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in Arabidopsis thaliana.

Microscopically visible chromatin is partitioned into two major components in Arabidopsis thaliana nuclei. On one hand, chromocenters are conspicuous foci of highly condensed "heterochromatic" domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich "euchromatin" emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes Arabidopsis a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, i.e., the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration.

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

Differential Regulation of Cyclin E by Yorkie-Scalloped Signaling in Organ Development.

Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. Here, we report a role of a growth modulator Yorkie (Yki), the Drosophila homolog of Yes-associated protein (YAP), that differentially regulates its targets in Drosophila wing imaginal discs; whereby Yki interacts with its transcriptional partner, Scalloped (Sd), the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell cycle regulator Cyclin E (CycE). Interestingly, when Yki was coexpressed with Fizzy-related (Fzr), a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells displayed larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression was not detected in the wing disc, coexpression of Fzr and Yki did not cause size differences between cells along the proximal-distal axis of the disc. We show that ectopic CycE promoted cell proliferation and apoptosis, and inhibited transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis.

Regulation of mitotic cytoskeleton dynamics and cytokinesis by integrin-linked kinase in retinoblastoma cells.

During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule dynamics and centrosome clustering, processes involved in cell cycle progression, and malignant transformation. In this study, we examine the effects of downregulating ILK on mitotic function in human retinoblastoma cell lines. These retinal cancer cells, caused by the loss of function of two gene alleles (Rb1) that encode the retinoblastoma tumour suppressor, have elevated expression of ILK. Here we show that inhibition of ILK activity results in a concentration-dependent increase in nuclear area and multinucleated cells. Moreover, inhibition of ILK activity and expression increased the accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical F-actin and centrosome de-clustering. Centrosome de-clustering, induced by ILK siRNA, was rescued in FLAG-ILK expressing Y79 cells as compared to those expressing FLAG-tag alone. Inhibition of ILK increased the proportion of cells exhibiting mitotic spindles and caused a significant G2/M arrest as early as 24 hours after exposure to QLT-0267. Live cell analysis indicate ILK downregulation causes an increase in multipolar anaphases and failed cytokinesis (bipolar and multipolar) of viable cells. These studies extend those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a novel role for ILK in cytokinesis of Rb deficient cells.

A novel transgenic mouse model carrying human Tribbles related protein 3 (TRB3) gene and its site specific phenotype.

Tribbles related protein 3 (TRB3) pseudokinase plays a crucial role in cell proliferation, migration and morphogenesis during development. In our recent study, an introduction of human TRB3 gene into mouse mammary tumor cells caused an increase of proliferation of tumor cells and their nuclear size. In the current study, to examine whether this gene causes de novo morphological changes in a specific organ site we have developed a novel variation of the transgenic mouse model that conditionally expresses human TRB3 (hTRB3) gene using Cre-recombinase (Cre)/loxP recombination system. By injecting hTRB3 transgene construct into pronuclei of mouse embryo, we eventually obtained four hTRB3 mice. The gene expression was controlled by infection of adenovirus-expressing Cre via the tail vein of hTRB3 mouse. In Cre-mediated hTRB3 mouse, expression of the hTRB3 protein was detected in the cytoplasm of hepatocytes in the liver. Expression of this protein was also seen in lymphocytes in the spleen, glomerular endothelial cells, and epithelial cells of collecting duct of the kidney. In hepatocytes of the hTRB3 mouse, nuclear size was significantly greater than that of the wild type mouse, indicating that hTRB3 can play a role at least in part in hepatic morphogenesis. The present animal model may provide a system for evaluation of de novo morphological changes induced by a specific transgene in a specific organ site.

Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes.

The quest to understand how the mechanical and geometrical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical and simple cell culture plate and the biological reality of actual tissue. In tissues, cells have their physical space constrained by neighboring cells and the extracellular matrix. Here, we propose a simple and versatile device to precisely and dynamically control this confinement parameter in cultured cells. We show that there is a precise threshold deformation above which the nuclear lamina breaks and reconstructs, whereas nuclear volume changes. We also show that different nuclear deformations correlate with the expression of specific sets of genes, including nuclear factors and classical mechanotransduction pathways. This versatile device thus enables the precise control of cell and nuclear deformation by confinement and the correlative study of the associated molecular events.

Disease modeling using embryonic stem cells: MeCP2 regulates nuclear size and RNA synthesis in neurons.

Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice.

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear:cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice.