Linker Of Nucleoskeleton Research Articles

Consequences of Lmna Exon 4 Mutations in Myoblast Function.

Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.

The testis-specific LINC component SUN3 is essential for sperm head shaping during mouse spermiogenesis

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome-manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain-containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Spatiotemporal Mislocalization of Nuclear Membrane-Associated Proteins in γ-Irradiation-Induced Senescent Cells.

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.

SUN1/2 Are Essential for RhoA/ROCK-Regulated Actomyosin Activity in Isolated Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.

Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is responsible for tethering the nucleus to the cytoskeleton, providing a pathway for the cell's nucleus to sense mechanical signals from the environment. Recently, we explored the role of the LINC complex in the development of glandular epithelial acini, such as those found in kidneys, breasts, and other organs. Acini developed with disrupted LINC complexes exhibited a loss of structural integrity, including filling of the lumen structures. As part of our investigation, we performed a mechanical indentation assay of LINC disrupted and undisrupted MDCK II cells using a micro-indentation instrument mounted above a laser-scanning confocal microscope. Through a combination of force measurements acquired from the micro-indentation instrument and contact area measurements taken from fluorescence images, we determined the average contact pressure at which the acini structure ruptured. Here, we provide a detailed description of the design of the micro-indentation instrument, as well as the experimental steps developed to perform these bio-indentation measurements. Furthermore, we discuss the data analysis steps necessary to determine the rupture pressure of the acini structures. While this protocol is focused on the indentation of individual glandular acini, the methods presented here can be adapted to perform a variety of mechanical indentation experiments for both 2D and 3D biological systems.

LINC-complex mediated positioning of the vegetative nucleus is involved in calcium and ROS signaling in Arabidopsis pollen tubes

ABSTRACT Nuclear movement and positioning play a role in developmental processes throughout life. Nuclear movement and positioning are mediated primarily by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes are comprised of the inner nuclear membrane SUN proteins and the outer nuclear membrane (ONM) KASH proteins. In Arabidopsis pollen tubes, the vegetative nucleus (VN) maintains a fixed distance from the pollen tube tip during growth, and the VN precedes the sperm cells (SCs). In pollen tubes of wit12 and wifi, mutants deficient in the ONM component of a plant LINC complex, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Subsequently, pollen tubes frequently fail to burst upon reception. In this study, we sought to determine if the pollen tube reception defect observed in wit12 and wifi is due to decreased sensitivity to reactive oxygen species (ROS). Here, we show that wit12 and wifi are hyposensitive to exogenous H2O2, and that this hyposensitivity is correlated with decreased proximity of the VN to the pollen tube tip. Additionally, we report the first instance of nuclear Ca2+ peaks in growing pollen tubes, which are disrupted in the wit12 mutant. In the wit12 mutant, nuclear Ca2+ peaks are reduced in response to exogenous ROS, but these peaks are not correlated with pollen tube burst. This study finds that VN proximity to the pollen tube tip is required for both response to exogenous ROS, as well as internal nuclear Ca2+ fluctuations.

The functional importance of lamins, actin, myosin, spectrin and the LINC complex in DNA repair.

Three major proteins in the nucleoskeleton, lamins, actin, and spectrin, play essential roles in maintenance of nuclear architecture and the integrity of the nuclear envelope, in mechanotransduction and mechanical coupling between the nucleoskeleton and cytoskeleton, and in nuclear functions such as regulation of gene expression, transcription and DNA replication. Less well known, but critically important, are the role these proteins play in DNA repair. The A-type and B-type lamins, nuclear actin and myosin, spectrin and the LINC (linker of nucleoskeleton and cytoskeleton) complex each function in repair of DNA damage utilizing various repair pathways. The lamins play a role in repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) or homologous recombination (HR). Actin is involved in repair of DNA DSBs and interacts with myosin in facilitating relocalization of these DSBs in heterochromatin for HR repair. Nonerythroid alpha spectrin (αSpII) plays a critical role in repair of DNA interstrand cross-links (ICLs) where it acts as a scaffold in recruitment of repair proteins to sites of damage and is important in the initial damage recognition and incision steps of the repair process. The LINC complex contributes to the repair of DNA DSBs and ICLs. This review will address the important functions of these proteins in the DNA repair process, their mechanism of action, and the profound impact a defect or deficiency in these proteins has on cellular function. The critical roles of these proteins in DNA repair will be further emphasized by discussing the human disorders and the pathophysiological changes that result from or are related to deficiencies in these proteins. The demonstrated function for each of these proteins in the DNA repair process clearly indicates that there is another level of complexity that must be considered when mechanistically examining factors crucial for DNA repair.Impact statementProteins in the nucleoskeleton, lamins, actin, myosin, and spectrin, have been shown to play critical roles in DNA repair. Deficiencies in these proteins are associated with a number of disorders. This review highlights the role these proteins and their association with the LINC complex play in DNA repair processes, their mechanism of action and the impacts deficiencies in these proteins have on DNA repair and on disorders associated with a deficiency in these proteins. It will clarify how these proteins, which interact with “classic DNA repair proteins” (e.g., RAD51, XPF), represent another level of complexity in the DNA repair process, which must be taken into consideration when carrying out mechanistic studies on proteins involved in DNA repair and in developing models for DNA repair pathways. This knowledge is essential for determining how deficiencies in these proteins relate to disorders resulting from loss of functional activity of these proteins.

Gene expression modulation by the linker of nucleoskeleton and cytoskeleton complex contributes to proteostasis.

Cellular mechanisms that act in concert to maintain protein homeostasis (proteostasis) are vital for organismal functionality and survival. Nevertheless, subsets of aggregation‐prone proteins form toxic aggregates (proteotoxicity) that in some cases, underlie the development of neurodegenerative diseases. Proteotoxic aggregates are often deposited in the vicinity of the nucleus, a process that is cytoskeleton‐dependent. Accordingly, cytoskeletal dysfunction contributes to pathological hallmarks of various neurodegenerative diseases. Here, we asked whether the linker of nucleoskeleton and cytoskeleton (LINC) complex, which bridges these filaments across the nuclear envelope, is needed for the maintenance of proteostasis. Employing model nematodes, we discovered that knocking down LINC components impairs the ability of the worm to cope with proteotoxicity. Knocking down anc‐1, which encodes a key component of the LINC complex, modulates the expression of transcription factors and E3 ubiquitin ligases, thereby affecting the rates of protein ubiquitination and impairing proteasome‐mediated protein degradation. Our results establish a link between the LINC complex, protein degradation, and neurodegeneration‐associated proteotoxicity.

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

A network of nuclear envelope proteins and cytoskeletal force generators mediates movements of and within nuclei throughout Caenorhabditis elegans development.

Nuclear migration and anchorage, together referred to as nuclear positioning, are central to many cellular and developmental events. Nuclear positioning is mediated by a conserved network of nuclear envelope proteins that interacts with force generators in the cytoskeleton. At the heart of this network are linker of nucleoskeleton and cytoskeleton (LINC) complexes made of Sad1 and UNC-84 (SUN) proteins at the inner nuclear membrane and Klarsicht, ANC-1, and Syne homology (KASH) proteins in the outer nuclear membrane. LINC complexes span the nuclear envelope, maintain nuclear envelope architecture, designate the surface of nuclei distinctly from the contiguous endoplasmic reticulum, and were instrumental in the early evolution of eukaryotes. LINC complexes interact with lamins in the nucleus and with various cytoplasmic KASH effectors from the surface of nuclei. These effectors regulate the cytoskeleton, leading to a variety of cellular outputs including pronuclear migration, nuclear migration through constricted spaces, nuclear anchorage, centrosome attachment to nuclei, meiotic chromosome movements, and DNA damage repair. How LINC complexes are regulated and how they function are reviewed here. The focus is on recent studies elucidating the best-understood network of LINC complexes, those used throughout Caenorhabditis elegans development.Impact statementDefects in nuclear positioning disrupt development in many mammalian tissues. In human development, LINC complexes play important cellular functions including nuclear positioning, homolog pairing in meiosis, DNA damage repair, wound healing, and gonadogenesis. The topics reviewed here are relevant to public health because defects in nuclear positioning and mutations in LINC components are associated with a wide variety of human diseases including muscular dystrophies, neurological disorders, progeria, aneurysms, hearing loss, blindness, sterility, and multiple cancers. Although this review focuses on findings in the model nematode Caenorhabditis elegans, the studies are relevant because almost all the findings originally made in C. elegans are conserved to humans. Furthermore, C. elegans remains the best described network for how LINC complexes are regulated and function.

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.

Characterization of 3D Printed Stretching Devices for Imaging Force Transmission in Live-Cells.

Cell stretch is a method which can rapidly apply mechanical force through cell-matrix and cell-cell adhesions and can be utilized to better understand underlying biophysical questions related to intracellular force transmission and mechanotransduction. 3D printable stretching devices suitable for live-cell fluorescent imaging were designed using finite element modeling and validated experimentally. These devices were then used along with FRET based nesprin-2G force sensitive biosensors as well as live cell fluorescent staining to understand how the nucleus responds to externally applied mechanical force in cells with both intact LINC (linker of nucleoskeleton and cytoskeleton) complex and cells with the LINC complex disrupted using expression of dominant negative KASH protein. The devices were shown to provide a larger strain ranges (300% uniaxial and 60% biaxial) than currently available commercial or academic designs we are aware of. Under uniaxial deformation, the deformation of the nucleus of NIH 3T3 cells per unit of imposed cell strain was shown to be approximately 50% higher in control cells compared to cells with a disrupted LINC complex. Under biaxial deformation, MDCK II cells showed permanent changes in the nuclear morphology as well as actin organization upon unloading, indicating that failure, plastic deformation, or remodeling of the cytoskeleton is occurring in response to the applied stretch. Development and open distribution of low-cost, 3D-printable uniaxial and biaxial cell stretching devices compatible with live-cell fluorescent imaging allows a wider range of researchers to investigate mechanical influences on biological questions with only a minimal investment of resources.

Abstract 436: Prediction of aggressive disease in metastatic castration-resistant prostate cancer: The role of very small nuclear circulating tumor cells

Abstract Background: Circulating tumor cells (CTCs) have arisen as a contemporary noninvasive prognostic biomarker for prostate cancer (PC). Previously, a subgroup of PC CTCs, with particularly small nuclei (<8.54 μm), were found to be correlated with poor prognosis and the emergence of visceral metastases (VM). This subgroup was named very-small nuclear CTCs (vsnCTCs). We proposed vsnCTCs as a biomarker of aggressive disease in metastatic castration-resistant PC (mCRPC). Previous Studies also showed that the disruption of emerin, a linker of nucleoskeleton and cytoskeleton complex proteins, increases the cancer cells' capacity of migration and invasion. We hypothesize that emerin mislocalization is potentially associated with vsnCTC formation and may be a critical step for metastasis. Methods: 35 blood samples were obtained from PC patients who failed first-line androgen deprivation therapy (ADT) and starting 2nd line treatment with abiraterone, enzalutamide, or taxane-based chemotherapy. Using our NanoVelcro CTC assay, we captured, subclassified, and enumerated the CTCs from patient samples with high-resolution imaging. Survival analyses were performed to exam the correlation between vsnCTC presence and patients’ prognosis. Concurrently, emerin staining was performed and the distribution and expression levels of emerin were analyzed in selected vsnCTC samples. Results: The presence of one or more vsnCTCs strongly correlated with inferior overall survival (OS), progression free survival (PFS), and time to VM (TTVM). We also observed lower emerin content in vsnCTCs compared to WBC, and more prominent emerin mislocalization in vsnCTCs compared to CTCs with larger nuclei. Conclusion: Our study strongly demonstrated the importance of morphologic characterization of CTCs and suggested that vsnCTC is a blood-borne biomarker for prediction of aggressive disease. Additionally, emerin mislocalization in vsnCTCs could be a potential biological pathway behind this morphologic phenomenon. Citation Format: Pin-Jung Chen, Yu Jen Jan, Pai-Chi Teng, Jie-Fu Chen, Shirley Cheng, Nu Yao, Mariana Reis-Sobreiro, Amber Lozano, Amy Gomez, Hsian-Rong Tseng, Michael Freeman, Edwin Posadas. Prediction of aggressive disease in metastatic castration-resistant prostate cancer: The role of very small nuclear circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 436.

Recovery of stem cell proliferation by low intensity vibration under simulated microgravity requires LINC complex

Mesenchymal stem cells (MSC) rely on their ability to integrate physical and spatial signals at load bearing sites to replace and renew musculoskeletal tissues. Designed to mimic unloading experienced during spaceflight, preclinical unloading and simulated microgravity models show that alteration of gravitational loading limits proliferative activity of stem cells. Emerging evidence indicates that this loss of proliferation may be linked to loss of cellular cytoskeleton and contractility. Low intensity vibration (LIV) is an exercise mimetic that promotes proliferation and differentiation of MSCs by enhancing cell structure. Here, we asked whether application of LIV could restore the reduced proliferative capacity seen in MSCs that are subjected to simulated microgravity. We found that simulated microgravity (sMG) decreased cell proliferation and simultaneously compromised cell structure. These changes included increased nuclear height, disorganized apical F-actin structure, reduced expression, and protein levels of nuclear lamina elements LaminA/C LaminB1 as well as linker of nucleoskeleton and cytoskeleton (LINC) complex elements Sun-2 and Nesprin-2. Application of LIV restored cell proliferation and nuclear proteins LaminA/C and Sun-2. An intact LINC function was required for LIV effect; disabling LINC functionality via co-depletion of Sun-1, and Sun-2 prevented rescue of cell proliferation by LIV. Our findings show that sMG alters nuclear structure and leads to decreased cell proliferation, but does not diminish LINC complex mediated mechanosensitivity, suggesting LIV as a potential candidate to combat sMG-induced proliferation loss.

SPAG4L/SPAG4L interacts with Nesprin2 to participate in the meiosis of spermatogenesis

SUN domain proteins are identified as a novel family of nuclear envelope proteins which are involved in spermatogenesis. SPAG4L is identified as the fifth member of this family. Previous studies have revealed that SPAG4L is involved in spermatogenesis and the mutations occurring in SPAG4L will lead to male infertility. However, the transcriptions of SPAG4L and its interacting proteins in the testis are still unclear. In this study, we identified a shorter transcript variant of SPAG4L, named SPAG4Lβ, in human testis by northern blot and reverse transcription-polymerase chain reaction. Bioinformatics analysis showed that it encodes a protein consisting of 311 amino acids, and subcellular localization analysis revealed that it is mainly expressed in the cytoplasm. In situ hybridization and immunofluorescence assay revealed that SPAG4L/SPAG4Lβ is involved in meiosis. Furthermore, co-IP results demonstrated that SPAG4L/SPAG4Lβ interacts with Nesprin2, a KASH domain protein to form the LINC (linker of nucleoskeleton and cytoskeleton) complexes. Immunofluorescence results revealed that the LINC complexes of Spag4l/Nesprin2 in mouse are involved in spermatocyte division. Our data indicated that SPAG4L/SPAG4Lβ may play an important role in the meiotic process.

Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression in primary mouse tumorigenic cells.

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

The Role of Nesprin in the Facial Branchiomotor Neuron (FBMN) Migration of Danio rerio (zebrafish) development

Nuclear envelope spectrin‐repeat proteins (Nesprins) are part of the LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, which plays a vital role in the binding of F‐actin cytoskeleton to the nuclear envelope. Nesprins are crucial in maintaining the integrity of the nuclear membrane. Nuclear membrane movement is a fundamental aspect in cell migration and muscle development. Previous work has demonstrated that Nesprins may be associated with myosin‐dependent tension, which has implications in muscular dystrophy due to the role that nesprins play in muscle cells. We hypothesize that expressing dominant‐negative nesprin in Danio rerio (zebrafish) embryos would result in defects in the migration of facial branchiomotor neurons and also cause disruptions in nuclear positioning in multinucleated muscle cells. To test this hypothesis, we use transient transgenic expression of dominant Nesprin in cranial motor neurons using the islet1 promoter. We quantify the extent of migration in neurons with inactive Nesprin. In addition, we express dominant‐negative Nesprin in muscle cells using a heat shock promoter and explore the positioning of nuclei in developing skeletal muscle myofibrils. Our preliminary data shows that transgenic zebrafish embryos with expression of dominant‐negative nesprin have disruptions and defects in tangential FBMN migration. However, with further experimentation and data collection in progress, we hope to analyze our findings for statistical significance. Our goal is to gain insight into the function of Nesprins in embryo development to better devise therapeutic strategies for patients with mutations in Nesprins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A noninvasive prognostic biomarker for metastatic castration-resistant prostate cancer: Very small nuclear circulating tumor cells.

179 Background: Circulating tumor cells (CTCs) have arisen as a contemporary biomarker for prostate cancer (PC). A subgroup of PC CTCs, with particularly small nuclei ( < 8.54 μm), were found to be correlated with poor prognosis and the emergence of visceral metastases (VM). This subgroup was named very-small nuclear CTCs (vsnCTCs). The findings led us to explore vsnCTCs as an aggressive biomarker in metastatic castration-resistant PC (mCRPC). We also explored a biological pathway that potentially drives this morphologic phenomenon. Studies showed that the disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, such as emerin, results in nuclear envelope instability and drives cancer cells to an amoeboid phenotype with increasing capacity of migration and invasion. We hypothesized that emerin mislocalization is associated with vsnCTC formation and may be a critical step of metastasis. Methods: Using our NanoVelcro CTC assay, we are able to capture and enumerate CTCs from patients' blood and correlate this data with clinical outcomes. We collected samples from 35 mCRPC patients who failed first-line androgen deprivation therapy and started treatment with abiraterone, enzalutamide, or taxane-based chemotherapy. Survival analyses were performed to exam the correlation between vsnCTC counts and patients’ prognosis. Concurrently, emerin staining was performed and the distribution and expression levels were analyzed in selected vsnCTC samples. Results: The presence of one or more vsnCTCs correlated with worse overall survival (P = 0.00013), progression free survival (PSA progression: P = 0.012; radiographic progression: P = 0.0015), and faster time to VM (P = 0.024). We also observed lower emerin content in vsnCTCs compared to WBC, and more prominent emerin mislocalization in vsnCTCs compared to CTCs with larger nuclei. Conclusions: Our study demonstrated the importance of morphologic characterization of CTCs and suggested that vsnCTCs is a putative biomarker for prediction of worse outcome. Additionally, our findings of emerin mislocalization in vsnCTCs suggested a potential biological pathway behind this nuclear morphologic phenomenon.

Signal Transduction across the Nuclear Envelope: Role of the LINC Complex in Bidirectional Signaling.

The primary functions of the nuclear envelope are to isolate the nucleoplasm and its contents from the cytoplasm as well as maintain the spatial and structural integrity of the nucleus. The nuclear envelope also plays a role in the transfer of various molecules and signals to and from the nucleus. To reach the nucleus, an extracellular signal must be transmitted across three biological membranes: the plasma membrane, as well as the inner and outer nuclear membranes. While signal transduction across the plasma membrane is well characterized, signal transduction across the nuclear envelope, which is essential for cellular functions such as transcriptional regulation and cell cycle progression, remains poorly understood. As a physical entity, the nuclear envelope, which contains more than 100 proteins, functions as a binding scaffold for both the cytoskeleton and the nucleoskeleton, and acts in mechanotransduction by relaying extracellular signals to the nucleus. Recent results show that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, which is a conserved molecular bridge that spans the nuclear envelope and connects the nucleoskeleton and cytoskeleton, is also capable of transmitting information bidirectionally between the nucleus and the cytoplasm. This short review discusses bidirectional signal transduction across the nuclear envelope, with a particular focus on mechanotransduction.

Desmin is Critical to the Nuclear Architecture of Cardiomyocytes

Long-term changes in mechanical stress on the heart are known to induce pathological remodeling, but the signaling pathways underlying the conversion of mechanical stress into transcriptional changes have not been established. Previous reports indicate that strain in the myocyte is transmitted to the nucleus at least in part by desmin, and it has been suggested that desmin may interact with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. Further, both gain and loss of function mutations in desmin are associated with the development of cardiomyopathy. Taken together, these observations raise questions about the role that desmin may play in myocyte sensing of and the transcriptional response to mechanical stress. To assess the role desmin plays in nuclear structure and transcriptional regulation, we acutely knocked down desmin using shRNA in adult cardiomyocytes. Initial fluorescence images indicate that desmin knockdown leads to a decrease in nuclear volume and large rearrangements (wrinkling) of the nuclear lamina. Electron microscopy on nuclei in these myocytes reveals dramatic nuclear structural changes following desmin knockdown. The disruptions of the lamina accompany large scale inward folding of the nuclear membrane, more than tripling membrane concave area. Additionally, the electron-dense chromatin regions typically associated with perilaminar heterochromatic domains expand to larger areas reaching, in many cases, hundreds of nanometers deeper into the interior of the nucleus. We also note substantial transcriptional and epigenetic changes resulting from desmin knockdown. These results suggest that desmin plays a critical role in maintaining nuclear architecture, and could form a link between mechanical stimuli acting on cardiomyocytes and their transcriptional results in the nucleus.