A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents.

Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope (NE) proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor, and SUN1 early in telophase, after the NE has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of NE proteins. Further, distinct mistargeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of NE formation in mammalian cells, and 3) distinct requirements of individual NE residents for continued targeting during the expansion phase of NE reformation.

Phosphorylation of the small heat shock protein HspB1 regulates cytoskeletal recruitment and cell motility.

The small heat shock protein HspB1, also known as Hsp25/27, is a ubiquitously expressed molecular chaperone that responds to mechanical cues. Uniaxial cyclic stretch activates the p38 mitogen-activated protein kinase (MAPK) signaling cascade and increases the phosphorylation of HspB1. Similar to the mechanosensitive cytoskeletal regulator zyxin, phospho-HspB1 is recruited to features of the stretch-stimulated actin cytoskeleton. To evaluate the role of HspB1 and its phosphoregulation in modulating cell function, we utilized CRISPR/Cas9-edited HspB1-null cells and determined they were altered in behaviors such as actin cytoskeletal remodeling, cell spreading, and cell motility. In our model system, expression of WT HspB1, but not nonphosphorylatable HspB1, rescued certain characteristics of the HspB1-null cells including the enhanced cell motility of HspB1-null cells and the deficient actin reinforcement of stretch-stimulated HspB1-null cells. The recruitment of HspB1 to high-tension structures in geometrically constrained cells, such as actin comet tails emanating from focal adhesions, also required a phosphorylatable HspB1. We show that mechanical signals activate posttranslational regulation of the molecular chaperone, HspB1, and are required for normal cell behaviors including actin cytoskeletal remodeling, cell spreading, and cell migration.

Nuclear pore complexes concentrate on Actin/LINC/Lamin nuclear lines in response to mechanical stress in a SUN1 dependent manner.

Formation of robust actomyosin stress fibers (SF) in response to cell stretch plays a key role in the transfer of information from the cytoplasm into the nucleus. Actin/LINC/Lamin (ALL) nuclear lines provide mechanical linkage between the actin cytoskeleton and the lamin nucleoskeleton across the nuclear envelope. To understand the establishment of ALL lines, we used live cell imaging of cells exposed to cyclic stretch. We discovered that nuclear pore complexes (NPCs) concentrate along ALL lines that are generated in response to uniaxial cyclic stretch. The ALL-associated NPCs display increased fluorescence intensity of nucleoporins Pom121, TPR and Nup153 relative to nucleoporins that are distal to the ALL lines. Here we test the hypothesis that a LINC complex component of ALL lines, SUN1 is involved in the integration of NPCs with ALL lines. We generated CRISPR SUN1 knockdown and knockout cell lines and show that SUN1 is essential for normal integration of NPCs to ALL lines. Loss or elimination of SUN1 significantly diminishes NPC/ALL line integration, demonstrating a key role for SUN1 in the recruitment or stabilization of NPCs to a discrete subdomain of the nuclear envelope at ALL lines. This work provides new insight into the mechanism by which cells respond to mechanical force through nuclear envelope remodeling.

Mechanical stress triggers nuclear remodeling and the formation of transmembrane actin nuclear lines with associated nuclear pore complexes.

Mechanical stimulation of fibroblasts induces changes in the actin cytoskeleton including stress fiber (SF) reinforcement and realignment. Here we characterize the nuclear response to mechanical stimulation (uniaxial cyclic stretch). Using fluorescence microscopy and quantitative image analysis we find that stretch-induced nuclear elongation and alignment perpendicular to the stretch vector are dependent on formin-regulated actin polymerization. The mechanosensitive transcription factors Yes-associated protein/Transcriptional coactivator with PDZ domain (YAP/TAZ) and myocardin-related transcription factor (MRTF-A, also known as MKL1 and MAL1) accumulate in the nucleus and activate their target genes in response to uniaxial cyclic stretch. We show that transmembrane actin nuclear (TAN) lines are induced by stretch stimulation and nuclear envelope (NE) proteins including nesprins, SUN2, and lamins form Linkers of the Nucleoskeleton and Cytoskeleton (LINC) complexes aligned with actin SFs. These NE structures are altered by pharmacological treatments (Cytochalasin D and Jasplakinolide) or genetic disruption (zyxin gene deletion) that alter actin, and their persistence requires maintenance of stretch stimulation. Nuclear pore complexes (NPCs) accumulate at TAN lines providing a potential mechanism for linking mechanical cues to NPC function.

Mechanical signals activate p38 MAPK pathway-dependent reinforcement of actin via mechanosensitive HspB1.

Despite the importance of a cell's ability to sense and respond to mechanical force, the molecular mechanisms by which physical cues are converted to cell-instructive chemical information to influence cell behaviors remain to be elucidated. Exposure of cultured fibroblasts to uniaxial cyclic stretch results in an actin stress fiber reinforcement response that stabilizes the actin cytoskeleton. p38 MAPK signaling is activated in response to stretch, and inhibition of p38 MAPK abrogates stretch-induced cytoskeletal reorganization. Here we show that the small heat shock protein HspB1 (hsp25/27) is phosphorylated in stretch-stimulated mouse fibroblasts via a p38 MAPK-dependent mechanism. Phosphorylated HspB1 is recruited to the actin cytoskeleton, displaying prominent accumulation on actin "comet tails" that emanate from focal adhesions in stretch-stimulated cells. Site-directed mutagenesis to block HspB1 phosphorylation inhibits the protein's cytoskeletal recruitment in response to mechanical stimulation. HspB1-null cells, generated by CRISPR/Cas9 nuclease genome editing, display an abrogated stretch-stimulated actin reinforcement response and increased cell migration. HspB1 is recruited to sites of increased traction force in cells geometrically constrained on micropatterned substrates. Our findings elucidate a molecular pathway by which a mechanical signal is transduced via activation of p38 MAPK to influence actin remodeling and cell migration via a zyxin-independent process.

The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics.

Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms-and their failure in asthma-remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

Molecular dissection of the mechanism by which EWS/FLI expression compromises actin cytoskeletal integrity and cell adhesion in Ewing sarcoma.

Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised.

LIM domains target actin regulators paxillin and zyxin to sites of stress fiber strain.

Contractile actomyosin stress fibers are critical for maintaining the force balance between the interior of the cell and its environment. Consequently, the actin cytoskeleton undergoes dynamic mechanical loading. This results in spontaneous, stochastic, highly localized strain events, characterized by thinning and elongation within a discrete region of stress fiber. Previous work showed the LIM-domain adaptor protein, zyxin, is essential for repair and stabilization of these sites. Using live imaging, we show paxillin, another LIM-domain adaptor protein, is also recruited to stress fiber strain sites. Paxillin recruitment to stress fiber strain sites precedes zyxin recruitment. Zyxin and paxillin are each recruited independently of the other. In cells lacking paxillin, actin recovery is abrogated, resulting in slowed actin recovery and increased incidence of catastrophic stress fiber breaks. For both paxillin and zyxin, the LIM domains are necessary and sufficient for recruitment. This work provides further evidence of the critical role of LIM-domain proteins in responding to mechanical stress in the actin cytoskeleton.

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators.

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.

The LIM Protein Zyxin Binds CARP-1 and Promotes Apoptosis.

Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation.

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling.

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement.

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

Targeted disruption of the murine zyxin gene.

Zyxin is an evolutionarily conserved protein that is concentrated at sites of cell adhesion, where it associates with members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of cytoskeletal regulators and is postulated to play a role in cytoskeletal dynamics and signaling. Zyxin transcripts are detected throughout murine embryonic development, and the protein is widely expressed in adults. Here we used a reverse genetic approach to examine the consequences of loss of zyxin function in the mouse. Mice that lack zyxin function are viable and fertile and display no obvious histological abnormalities in any of the organs examined. Because zyxin contributes to the localization of Ena/VASP family members at certain subcellular locations, we carefully examined the zyxin(-/-) mice for evidence of defects that have been observed when Ena/VASP proteins are compromised in the mouse. Specifically, we evaluated blood platelet function, nervous system development, and skin architecture but did not detect any defects in these systems. Zyxin is the founding member of a family of proteins that also includes the lipoma preferred partner (LPP) and thyroid receptor-interacting protein 6 (TRIP6). These zyxin family members display patterns of expression that significantly overlap that of zyxin. Western blot analysis indicates that there is no detectable upregulation of either LPP or TRIP6 expression in tissues derived from zyxin-null mice. Because zyxin family members may have overlapping functions, a comprehensive understanding of the role of these proteins in the mouse will require the generation of compound mutations in which multiple zyxin family members are simultaneously compromised.

Members of the Zyxin family of LIM proteins interact with members of the p130Cas family of signal transducers.

Integrin binding to extracellular matrix proteins induces formation of signaling complexes at focal adhesions. Zyxin co-localizes with integrins at sites of cell-substratum adhesion and is postulated to serve as a docking site for the assembly of multimeric protein complexes involved in regulating cell motility. Recently, we identified a new member of the zyxin family called TRIP6. TRIP6 is localized at focal adhesions and overexpression of TRIP6 slows cell migration. In an effort to define the molecular mechanism by which TRIP6 affects cell migration, the yeast two-hybrid assay was employed to identify proteins that directly bind to TRIP6. This assay revealed that both TRIP6 and zyxin interact with CasL/HEF1, a member of the Cas family. This association is mediated by the LIM region of the zyxin family members and the SH2 domain-binding region of CasL/HEF1. Furthermore, the association between p130(Cas) and the two zyxin family members was demonstrated to occur in vivo by co-immunoprecipitation. Zyxin and Cas family members may cooperate to regulate cell motility.