CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.     

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.     

Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts

In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.     

The role of the cell nucleus in mechanotransduction

Mechanical forces are known to influence cellular processes with consequences at the cellular and physiological level. The cell nucleus is the largest and stiffest organelle, and it is connected to the cytoskeleton for proper cellular function. The connection between the nucleus and the cytoskeleton is in most cases mediated by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Not surprisingly, the nucleus and the associated cytoskeleton are implicated in multiple mechanotransduction pathways important for cellular activities. Herein, we review recent advances describing how the LINC complex, the nuclear lamina, and nuclear pore complexes are involved in nuclear mechanotransduction. We will also discuss how the perinuclear actin cytoskeleton is important for the regulation of nuclear mechanotransduction. Additionally, we discuss the relevance of nuclear mechanotransduction for cell migration, development, and how nuclear mechanotransduction impairment leads to multiple disorders.     

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.     

The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.     

Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)-1 and -2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin alpha6beta4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.     

FHOD1 coordinates actin filament and microtubule alignment to mediate cell elongation

Diaphanous-related formins (DRFs) are actin nucleators that mediate rearrangements of the actin cytoskeleton downstream of specific Rho GTPases. The DRF Formin Homology 2 Domain containing 1 (FHOD1) interacts with the Rac1 GTPase and induces the formation of and associates with bundled actin stress fibers. Here we report that active FHOD1 also coordinates microtubules with these actin stress fibers. Expression of a constitutive active FHOD1 variant in HeLa cells not only resulted in pronounced formation of FHOD1-actin fibers but also caused marked cell elongation and parallel alignment of microtubules without affecting cytokinesis of these cells. The analysis of deletions in the FH1 and FH2 functional regions revealed that the integrity of both domains was strictly required for FHOD1's effects on the cytoskeleton. Dominant-negative approaches demonstrated that filament coordination and cell elongation depended on the activity of the Rho-ROCK cascade, but did not involve Rac or Cdc42 activity. Experimental depolymerization of actin filaments or microtubules revealed that the formation of FHOD1-actin fibers was a prerequisite for the polarization of microtubules. However, only simultaneous disruption of both filament systems reversed the cell elongation induced by activated FHOD1. Thus, sustained cell elongation was a consequence of FHOD1-mediated actin-microtubule coordination. These results suggest filament coordination as a conserved function of mammalian DRFs.     

Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.     

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape.     

Actomyosin Tension Exerted on the Nucleus through Nesprin-1 Connections Influences Endothelial Cell Adhesion, Migration, and Cyclic Strain-Induced Reorientation

Endothelial cell polarization and directional migration is required for angiogenesis. Polarization and motility requires not only local cytoskeletal remodeling but also the motion of intracellular organelles such as the nucleus. However, the physiological significance of nuclear positioning in the endothelial cell has remained largely unexplored. Here, we show that siRNA knockdown of nesprin-1, a protein present in the linker of nucleus to cytoskeleton complex, abolished the reorientation of endothelial cells in response to cyclic strain. Confocal imaging revealed that the nuclear height is substantially increased in nesprin-1 depleted cells, similar to myosin inhibited cells. Nesprin-1 depletion increased the number of focal adhesions and substrate traction while decreasing the speed of cell migration; however, there was no detectable change in nonmuscle myosin II activity in nesprin-1 deficient cells. Together, these results are consistent with a model in which the nucleus balances a portion of the actomyosin tension in the cell. In the absence of nesprin-1, actomyosin tension is balanced by the substrate, leading to abnormal adhesion, migration, and cyclic strain-induced reorientation.     

A role for nuclear envelope–bridging complexes in homology-directed repair

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

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.     

Important but differential roles for actin in trafficking of Epstein-Barr virus in B cells and epithelial cells

Epstein-Barr virus (EBV) uses different virus and cell proteins to enter its two major targets, B lymphocytes and epithelial cells. The routes that the virus takes into the two cell types are also different. To determine if these differences extend to movement from the cell surface to the nucleus, we examined the fate of incoming virus. Essentially all virus that entered a B cell remained stable for at least 8 h. In contrast, up to 80% of virus entering an epithelial cell was degraded in a compartment sensitive to inhibitors of components involved in autophagy. Inhibitors of actin remodeling blocked entry into a B cell but had no effect or enhanced entry into an epithelial cell. Inhibitors of the microtubule network reduced intracellular transport in both cell types, but movement to the nucleus in an epithelial cell also required involvement of the actin cytoskeleton. Deletion of the cytoplasmic tail of CR2, which in an epithelial cell interacts with the actin nucleator FHOS/FHOD when cross-linked by EBV, had no effect on infection. However, inhibitors of downstream signaling by integrins reduced intracellular transport. Cooperation of the microtubule and actin cytoskeletons, possibly activated by interaction with integrin binding proteins in the envelope of EBV, is needed for successful infection of an epithelial cell.     

Nuclear mechanotransduction in stem cells

In development and in homeostatic maintenance of tissues, stem cells and progenitor cells are constantly subjected to forces. These forces can lead to significant changes in gene expression and function of stem cells, mediating self-renewal, lineage specification, and even loss of function. One of the ways that has been proposed to mediate these functional changes in stem cells is nuclear mechanotransduction — the process by which forces are converted to signals in the nucleus. The purpose of this review is to discuss the means by which mechanical signals are transduced into the nucleus, through the linker of nucleoskeleton and cytoskeleton (LINC) complex and other nuclear envelope transmembrane (NET) proteins, which connect the cytoskeleton to the nucleus. We discuss how LINC/NETs confers tissue-specific mechanosensitivity to cells and further elucidate how LINC/NETs acts as a control center for nuclear mechanical signals, regulating both gene expression and chromatin organization. Throughout, we primarily focus on stem cell–specific examples, notwithstanding that this is a nascent field. We conclude by highlighting open questions and pointing the way to enhanced research efforts to understand the role nuclear mechanotransduction plays in cell fate choice.