LINC complex protein nesprin-2 has pro-apoptotic activity via Bcl-2 family proteins.

The apoptotic intrinsic pathway is initiated by perforation of the mitochondrial outer membrane by the effector pro-apoptotic proteins of the Bcl-2 family, Bax and Bak. Bax and Bak need to be activated, a process facilitated by the action of BH3-only pro-apoptotic members of the Bcl-2 family. The latter either directly activates the effector proteins or antagonizes the action of pro-survival Bcl-2 family members such as Bcl-xL. The nuclear envelope is a known target of the apoptotic machinery; however, it may also act as mediator of apoptosis. We showed previously that the nuclear envelope protein nesprin-2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex, can bind to Bax in close proximity to the mitochondria and that the binding increases in apoptotic cells. We now show that depleting nesprin-2 inhibits the apoptotic mitochondrial pathway as measured by Bax and Bak activation and cytochrome c release. This survival effect was Bcl-xL-dependent. Nesprin-2 depletion also inhibited spontaneous exposure of the N-terminus of Bak in cells lacking Bcl-xL and increased the presence of Bcl-xL and Bax in the mitochondria. These results indicate that nesprin-2 promotes Bak activation and regulates mitochondrial translocation/retrotranslocation of Bcl-2 family proteins. Our findings demonstrate a new apoptotic pathway whereby the nuclear envelope, via nesprin-2, regulates apoptosis.

FHODs: Nuclear tethered formins for nuclear mechanotransduction.

In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and FHOD3. Their similar biochemical properties suggest overlapping and redundant functions. FHOD1 is widely expressed, FHOD3 less so, with highest expression in skeletal (FHOD1) and cardiac (FHOD3) muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple FHOD3 variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to "hot spots" in FHOD3 domains. We discuss how FHOD1/3's role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle.

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics.

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin­LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins.

The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins.

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

ERK1/2 Phosphorylation of FHOD Connects Signaling and Nuclear Positioning Alternations in Cardiac Laminopathy.

Mutations in the lamin A/C gene (LMNA) cause cardiomyopathy and also disrupt nuclear positioning in fibroblasts. LMNA mutations causing cardiomyopathy elevate ERK1/2 activity in the heart, and inhibition of the ERK1/2 kinase activity ameliorates pathology, but the downstream effectors remain largely unknown. We now show that cardiomyocytes from mice with an Lmna mutation and elevated cardiac ERK1/2 activity have altered nuclear positioning. In fibroblasts, ERK1/2 activation negatively regulated nuclear movement by phosphorylating S498 of FHOD1. Expression of an unphosphorylatable FHOD1 variant rescued the nuclear movement defect in fibroblasts expressing a cardiomyopathy-causing lamin A mutant. In hearts of mice with LMNA mutation-induced cardiomyopathy, ERK1/2 mediated phosphorylation of FHOD3, an isoform highly expressed in cardiac tissue. Phosphorylation of FHOD1 and FHOD3 inhibited their actin bundling activity. These results show that phosphorylation of FHOD proteins by ERK1/2 is a critical switch for nuclear positioning and may play a role in the pathogenesis of cardiomyopathy caused by LMNA mutations.

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.

Imbalanced nucleocytoskeletal connections create common polarity defects in progeria and physiological aging.

Studies of the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) can potentially reveal cellular defects associated with physiological aging. HGPS results from expression and abnormal nuclear envelope association of a farnesylated, truncated variant of prelamin A called "progerin." We surveyed the diffusional mobilities of nuclear membrane proteins to identify proximal effects of progerin expression. The mobilities of three proteins-SUN2, nesprin-2G, and emerin-were reduced in fibroblasts from children with HGPS compared with those in normal fibroblasts. These proteins function together in nuclear movement and centrosome orientation in fibroblasts polarizing for migration. Both processes were impaired in fibroblasts from children with HGPS and in NIH 3T3 fibroblasts expressing progerin, but were restored by inhibiting protein farnesylation. Progerin affected both the coupling of the nucleus to actin cables and the oriented flow of the cables necessary for nuclear movement and centrosome orientation. Progerin overexpression increased levels of SUN1, which couples the nucleus to microtubules through nesprin-2G and dynein, and microtubule association with the nucleus. Reducing microtubule-nuclear connections through SUN1 depletion or dynein inhibition rescued the polarity defects. Nuclear movement and centrosome orientation were also defective in fibroblasts from normal individuals over 60 y, and both defects were rescued by reducing the increased level of SUN1 in these cells or inhibiting dynein. Our results identify imbalanced nuclear engagement of the cytoskeleton (microtubules: high; actin filaments: low) as the basis for intrinsic cell polarity defects in HGPS and physiological aging and suggest that rebalancing the connections can ameliorate the defects.

Mechanical principles of nuclear shaping and positioning.

Positioning and shaping the nucleus represents a mechanical challenge for the migrating cell because of its large size and resistance to deformation. Cells shape and position the nucleus by transmitting forces from the cytoskeleton onto the nuclear surface. This force transfer can occur through specialized linkages between the nuclear envelope and the cytoskeleton. In response, the nucleus can deform and/or it can move. Nuclear movement will occur when there is a net differential in mechanical force across the nucleus, while nuclear deformation will occur when mechanical forces overcome the mechanical resistance of the various structures that comprise the nucleus. In this perspective, we review current literature on the sources and magnitude of cellular forces exerted on the nucleus, the nuclear envelope proteins involved in transferring cellular forces, and the contribution of different nuclear structural components to the mechanical response of the nucleus to these forces.

Analysis of Nesprin-2 Interaction with Its Binding Partners and Actin.

Nuclei are connected to the actin cytoskeleton for controlling its position in the cell and for mechanochemical signaling. Nesprin-2G is one of the major outer nuclear membrane proteins that links the nucleus to the actin cytoskeleton. In addition to its paired calponin homology (CH) domains, nesprin-2G interacts with actin filaments by binding the actin-bundling proteins FHOD1 and fascin. We describe methods to measure the interaction of nesprin-2G with actin filaments using an actin co-sedimentation assay and with its binding partner FHOD1 using a GST pull-down method.

Centrifugal Displacement of Nuclei in Adherent Cells to Study LINC Complex-Dependent Mechanisms of Homeostatic Nuclear Positioning.

The positioning of the nucleus is critical for key cellular processes including division, migration, and differentiation. Traditional approaches to understanding the functions and mechanisms of nuclear positioning have relied upon cellular systems in which nuclei move in response to stimuli or developmental programs and use molecular or pharmacological perturbations of nuclear and cytoskeletal elements. Here, we describe a complimentary approach to perturbing nuclear position in adherent cells using centrifugal force and how this may be used to understand LINC complex mechanisms of homeostatic nuclear positioning.

Nuclear ARP2/3 drives DNA break clustering for homology-directed repair.

DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.

Nuclear positioning in migrating fibroblasts.

The positioning and movement of the nucleus has recently emerged as an important aspect of cell migration. Understanding of nuclear positioning and movement has reached an apogee in studies of fibroblast migration. Specific nuclear positioning and movements have been described in the polarization of fibroblast for cell migration and in active migration in 2D and 3D environments. Here, we review recent studies that have uncovered novel molecular mechanisms that contribute to these events in fibroblasts. Many of these involve a connection between the nucleus and the cytoskeleton through the LINC complex composed of outer nuclear membrane nesprins and inner nuclear membrane SUN proteins. We consider evidence that appropriate nuclear positioning contributes to efficient fibroblast polarization and migration and the possible mechanism through which the nucleus affects cell migration.

Centrifugal Displacement of Nuclei Reveals Multiple LINC Complex Mechanisms for Homeostatic Nuclear Positioning.

Nuclear movement is critical for developmental events, cell polarity, and migration and is usually mediated by linker of nucleoskeleton and cytoskeleton (LINC) complexes connecting the nucleus to cytoskeletal elements. Compared to active nuclear movement, relatively little is known about homeostatic positioning of nuclei, including whether it is an active process. To explore homeostatic nuclear positioning, we developed a method to displace nuclei in adherent cells using centrifugal force. Nuclei displaced by centrifugation rapidly recentered by mechanisms that depended on cell context. In cell monolayers with wounds oriented orthogonal to the force, nuclei were displaced toward the front and back of the cells on the two sides of the wound. Nuclei recentered from both positions, but at different rates and with different cytoskeletal linkage mechanisms. Rearward recentering was actomyosin, nesprin-2G, and SUN2 dependent, whereas forward recentering was microtubule, dynein, nesprin-2G, and SUN1 dependent. Nesprin-2G engaged actin through its N terminus and microtubules through a novel dynein interacting site near its C terminus. Both activities were necessary to maintain nuclear position in uncentrifuged cells. Thus, even when not moving, nuclei are actively maintained in position by engaging the cytoskeleton through the LINC complex.

Stabilization of dynamic microtubules by mDia1 drives Tau-dependent Aß1-42 synaptotoxicity.

Oligomeric Amyloid ß1-42 (Aß) plays a crucial synaptotoxic role in Alzheimer's disease, and hyperphosphorylated tau facilitates Aß toxicity. The link between Aß and tau, however, remains controversial. In this study, we find that in hippocampal neurons, Aß acutely induces tubulin posttranslational modifications (PTMs) and stabilizes dynamic microtubules (MTs) by reducing their catastrophe frequency. Silencing or acute inhibition of the formin mDia1 suppresses these activities and corrects the synaptotoxicity and deficits of axonal transport induced by Aß. We explored the mechanism of rescue and found that stabilization of dynamic MTs promotes tau-dependent loss of dendritic spines and tau hyperphosphorylation. Collectively, these results uncover a novel role for mDia1 in Aß-mediated synaptotoxicity and demonstrate that inhibition of MT dynamics and accumulation of PTMs are driving factors for the induction of tau-mediated neuronal damage.

TorsinA controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

Identification of ginkgolide targets in brain by photoaffinity labeling.

Ginkgolides are terpene trilactones in Ginkgo biloba, a popular medicinal herb for memory disorders. Although ginkgolides are known for various neurobiological effects, their macromolecular target in brain is unknown. In this work, we employed benzophenone derivatives of ginkgolides to identify their binding target in brain. Photolabeling of bovine hippocampus homogenates identified a series of α-tubulin isotypes. Selective photolabeling of α-tubulin over ß-tubulin, which is equally abundant in brain, suggested that ginkgolides might modulate microtubule biology differently than typical microtubule-binding agents, such as taxol. In fact, ginkgolide A did not affect microtubule polymerization or cell proliferation; instead, it inhibited detyrosination of α-tubulin and reorientation of microtubule-organizing centers. Taken together, the current findings indicate that ginkgolides constitute a new class of microtubule-binding agents with distinct effects on α-tubulin biology.

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells.

Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

Swollen Nuclei Signal from the Grave.

Eicosanoid signaling plays key pro-inflammatory roles during tissue damage. Now, Enyedi et al. show that swelling of nuclei in cell corpses activates eicosanoid signaling to recruit leukocytes to sites of tissue damage. The enhanced membrane tension in swollen nuclei directly promotes calcium-dependent translocation and activation of enzymes involved in eicosanoid biosynthesis.