LINCking the Nuclear Envelope to Sperm Architecture.

Nuclear architecture undergoes an extensive remodeling during spermatogenesis, especially at levels of spermatocytes (SPC) and spermatids (SPT). Interestingly, typical events of spermiogenesis, such as nuclear elongation, acrosome biogenesis, and flagellum formation, need a functional cooperation between proteins of the nuclear envelope and acroplaxome/manchette structures. In addition, nuclear envelope plays a key role in chromosome distribution. In this scenario, special attention has been focused on the LINC (linker of nucleoskeleton and cytoskeleton) complex, a nuclear envelope-bridge structure involved in the connection of the nucleoskeleton to the cytoskeleton, governing mechanotransduction. It includes two integral proteins: KASH- and SUN-domain proteins, on the outer (ONM) and inner (INM) nuclear membrane, respectively. The LINC complex is involved in several functions fundamental to the correct development of sperm cells such as head formation and head to tail connection, and, therefore, it seems to be important in determining male fertility. This review provides a global overview of the main LINC complex components, with a special attention to their subcellular localization in sperm cells, their roles in the regulation of sperm morphological maturation, and, lastly, LINC complex alterations associated to male infertility.

Changes in Nuclear Shape and Gene Expression in Response to Simulated Microgravity Are LINC Complex-Dependent.

Microgravity is known to affect the organization of the cytoskeleton, cell and nuclear morphology and to elicit differential expression of genes associated with the cytoskeleton, focal adhesions and the extracellular matrix. Although the nucleus is mechanically connected to the cytoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, the role of this group of proteins in these responses to microgravity has yet to be defined. In our study, we used a simulated microgravity device, a 3-D clinostat (Gravite), to investigate whether the LINC complex mediates cellular responses to the simulated microgravity environment. We show that nuclear shape and differential gene expression are both responsive to simulated microgravity in a LINC-dependent manner and that this response changes with the duration of exposure to simulated microgravity. These LINC-dependent genes likely represent elements normally regulated by the mechanical forces imposed by gravity on Earth.

Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase.

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

Insight into Mechanobiology: How Stem Cells Feel Mechanical Forces and Orchestrate Biological Functions.

The cross-talk between stem cells and their microenvironment has been shown to have a direct impact on stem cells' decisions about proliferation, growth, migration, and differentiation. It is well known that stem cells, tissues, organs, and whole organisms change their internal architecture and composition in response to external physical stimuli, thanks to cells' ability to sense mechanical signals and elicit selected biological functions. Likewise, stem cells play an active role in governing the composition and the architecture of their microenvironment. Is now being documented that, thanks to this dynamic relationship, stemness identity and stem cell functions are maintained. In this work, we review the current knowledge in mechanobiology on stem cells. We start with the description of theoretical basis of mechanobiology, continue with the effects of mechanical cues on stem cells, development, pathology, and regenerative medicine, and emphasize the contribution in the field of the development of ex-vivo mechanobiology modelling and computational tools, which allow for evaluating the role of forces on stem cell biology.

Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex.

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.

Chain reaction: LINC complexes and nuclear positioning.

Nuclear positioning plays an essential role in defining cell architecture and behaviour in both development and disease, and nuclear location frequently adjusts according to internal and external cues. For instance, during periods of migration in many cell types, the nucleus may be actively repositioned behind the microtubule-organising centre. Nuclear movement, for the most part, is dependent upon coupling of the cytoskeleton to the nuclear periphery. This is accomplished largely through SUN and KASH domain proteins, which together assemble to form LINC (linker of the nucleoskeleton and cytoskeleton) complexes spanning the nuclear envelope. SUN proteins of the inner nuclear membrane provide a connection to nuclear structures while acting as a tether for outer nuclear membrane KASH proteins. The latter contain binding sites for diverse cytoskeletal components. Recent publications highlight new aspects of LINC complex regulation revealing that the interplay between SUN and KASH partners can strongly influence how the nucleus functionally engages with different branches of the cytoskeleton.

Physical Signals May Affect Mesenchymal Stem Cell Differentiation via Epigenetic Controls.

Marrow mesenchymal stem cells supply bone osteoblasts and adipocytes. Exercise effects to increase bone and decrease fat involve transfer of signals from the cytoplasm into the nucleus to regulate gene expression. We propose that exercise control of stem cell fate relies on structural connections that terminate in the nucleus and involve intranuclear actin structures that regulate epigenetic gene expression.

The Nucleoskeleton.

SUMMARYThe nucleoskeleton is an important structural feature of the metazoan nucleus and is involved in the regulation of genome expression and maintenance. The nuclear lamins are intermediate filament proteins that form a peripheral nucleoskeleton in concert with other lamin-associated proteins. Several other proteins normally found in the cytoskeleton have also been identified in the nucleus, but, as will be discussed here, their roles in forming a nucleoskeleton have not been elucidated. Nevertheless, mutations in lamins and lamin-associated proteins cause a spectrum of diseases, making them interesting targets for future research.

Nuclear Mechanics and Stem Cell Differentiation.

Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis.

Replication fork stalling at DNA lesions is a common problem during the process of DNA replication. One way to allow the bypass of these lesions is via specific recombination-based mechanisms that involve switching of the replication template to the sister chromatid. Inherent to these mechanisms is the formation of DNA joint molecules (JMs) between sister chromatids. Such JMs need to be disentangled before chromatid separation in mitosis and the activity of JM resolution enzymes, which is under stringent cell cycle control, is therefore up-regulated in mitosis. An additional layer of control is facilitated by scaffold proteins. In budding yeast, specifically during mitosis, Slx4 and Dpb11 form a cell cycle kinase-dependent complex with the Mus81-Mms4 structure-selective endonuclease, which allows efficient JM resolution by Mus81. Furthermore, Slx4 and Dpb11 interact even prior to joining Mus81 and respond to replication fork stalling in S-phase. This S-phase complex is involved in the regulation of the DNA damage checkpoint as well as in early steps of template switch recombination. Similar interactions and regulatory principles are found in human cells suggesting that Slx4 and Dpb11 may have an evolutionary conserved role organizing the cellular response to replication fork stalling.

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength.

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that Rad54B--a SNF2 helicase-like DNA-repair protein--limits the strength of both the G1/S and G2/M checkpoints. We find that Rad54B functions as a scaffold for p53 degradation via its direct interaction with the MDM2-MDMX ubiquitin-ligase complex. During the early phases of the DDR, Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the p53-mediated checkpoint is established, Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.

Linker of nucleoskeleton and cytoskeleton complex proteins in cardiac structure, function, and disease.

The linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of proteins within the inner and the outer nuclear membranes, connects the nuclear lamina to the cytoskeleton. The importance of this complex has been highlighted by the discovery of mutations in genes encoding LINC complex proteins, which cause skeletal or cardiac myopathies. Herein, this review summarizes structure, function, and interactions of major components of the LINC complex, highlights how mutations in these proteins may lead to cardiac disease, and outlines future challenges in the field.

Spatio-temporal organization of replication in bacteria and eukaryotes (nucleoids and nuclei).

Here we discuss the spatio-temporal organization of replication in eubacteria and eukaryotes. Although there are significant differences in how replication is organized in cells that contain nuclei from those that do not, you will see that organization of replication in all organisms is principally dictated by the structured arrangement of the chromosome. We will begin with how replication is organized in eubacteria with particular emphasis on three well studied model organisms. We will then discuss spatial and temporal organization of replication in eukaryotes highlighting the similarities and differences between these two domains of life.

Spatial proximity and similarity of the epigenetic state of genome domains.

Recent studies demonstrate that the organization of the chromatin within the nuclear space might play a crucial role in the regulation of gene expression. The ongoing progress in determination of the 3D structure of the nuclear chromatin allows one to study correlations between spatial proximity of genome domains and their epigenetic state. We combined the data on three-dimensional architecture of the whole human genome with results of high-throughput studies of the chromatin functional state and observed that fragments of different chromosomes that are spatially close tend to have similar patterns of histone modifications, methylation state, DNAse sensitivity, expression level, and chromatin states in general. Moreover, clustering of genome regions by spatial proximity produced compact clusters characterized by the high level of histone modifications and DNAse sensitivity and low methylation level, and loose clusters with the opposite characteristics. We also associated the spatial proximity data with previously detected chimeric transcripts and the results of RNA-seq experiments and observed that the frequency of formation of chimeric transcripts from fragments of two different chromosomes is higher among spatially proximal genome domains. A fair fraction of these chimeric transcripts seems to arise post-transcriptionally via trans-splicing.

The nucleoskeleton as a genome-associated dynamic 'network of networks'.

In the cytosol, actin polymers, intermediate filaments and microtubules can anchor to cell surface adhesions and interlink to form intricate networks. This cytoskeleton is anchored to the nucleus through LINC (links the nucleoskeleton and cytoskeleton) complexes that span the nuclear envelope and in turn anchor to networks of filaments in the nucleus. The metazoan nucleoskeleton includes nuclear pore-linked filaments, A-type and B-type lamin intermediate filaments, nuclear mitotic apparatus (NuMA) networks, spectrins, titin, 'unconventional' polymers of actin and at least ten different myosin and kinesin motors. These elements constitute a poorly understood 'network of networks' that dynamically reorganizes during mitosis and is responsible for genome organization and integrity.

A scaffold for X chromosome inactivation.

X chromosome inactivation (XCI), the silencing of one of the two X chromosomes in XX female cells, equalises the dosage of X-linked genes relative to XY males. The process is mediated by the non-coding RNA X inactive specific transcript (Xist) that binds in cis and propagates along the inactive X chromosome elect, triggering chromosome-wide silencing. The mechanisms by which Xist RNA binds and spreads along the chromosome, and initiates Xist-mediated chromosome silencing remain poorly understood. Accumulating evidence suggests that chromosome and nuclear organisation are important in both processes. Notably, recent studies have identified specific factors, previously shown to be components of the nuclear matrix or scaffold, to play a role both in Xist RNA-binding and in Xist-mediated silencing. In this review we provide a perspective on these studies in the context of previous work on chromosome/nuclear architecture in XCI.

Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina.

Although interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) are implicated in various nuclear functions, the understanding of the regulatory mechanism of MARs is still poor. A few MAR-binding proteins (MARBP) have been isolated from some plants and animals, but not from the unicellular algae. Here, we identify a novel MAR-binding protein, namely DMBP-1, from the halotolerant alga Dunaliella salina. The cDNA of DMBP-1 is 2322-bp long and contains a 1626 bp of an open reading frame encoding a polypeptide of 542 amino acids (59 kDa). The DMBP-1 expressed in Escherichia coli specifically binds A/T-rich MAR DNA. The DMBP-1 fused to green fluorescent protein appears only inside the nuclei of Chinese hamster ovarian cells transfected with the pEGFP-MBP, indicating that the protein is located in the nuclei. The findings mentioned above may contribute to better understanding of the nuclear matrix-MAR interactions.

Epigenetic modifications in 3D: nuclear organization of the differentiating mammary epithelial cell.

During the development of tissues, complex programs take place to reach terminally differentiated states with specific gene expression profiles. Epigenetic regulations such as histone modifications and chromatin condensation have been implicated in the short and long-term control of transcription. It has recently been shown that the 3D spatial organization of chromosomes in the nucleus also plays a role in genome function. Indeed, the eukaryotic interphase nucleus contains sub-domains that are characterized by their enrichment in specific factors such as RNA Polymerase II, splicing machineries or heterochromatin proteins which render portions of the genome differentially permissive to gene expression. The positioning of individual genes relative to these sub-domains is thought to participate in the control of gene expression as an epigenetic mechanism acting in the nuclear space. Here, we review what is known about the sub-nuclear organization of mammary epithelial cells in connection with gene expression and epigenetics. Throughout differentiation, global changes in nuclear architecture occur, notably with respect to heterochromatin distribution. The positions of mammary-specific genes relative to nuclear sub-compartments varies in response to hormonal stimulation. The contribution of tissue architecture to cell differentiation in the mammary gland is also seen at the level of nuclear organization, which is sensitive to microenvironmental stimuli such as extracellular matrix signaling. In addition, alterations in nuclear organization are concomitant with immortalization and carcinogenesis. Thus, the fate of cells appears to be controlled by complex pathways connecting external signal integration, gene expression, epigenetic modifications and chromatin organization in the nucleus.

Roles for nuclear organization in the maintenance of genome stability.

Recent findings demonstrate that chromatin dynamics and nuclear organization are not only important for gene regulation but also for the maintenance of genome stability. Thanks to novel techniques that allow the visualization of specific chromatin domains in living cells, recent studies have demonstrated that the spatial dynamics of double-strand breaks and modifying enzymes can influence repair. The importance of the spatial organization in the repair of DNA damage has been confirmed by demonstrating that perturbation of nuclear organization can lead to gene amplifications, deletions, translocations and end-to-end telomere fusion events.