Distinct Nuclear-Cytoskeletal LINCages Position the Nucleus for Homeostasis, Polarization and Migration

Nuclear positioning occurs in different cellular contexts: from dividing yeast to more specialized cells like neuronal glial progenitor and skeletal muscle cells. Interestingly, abnormal nuclear positioning is associated with diseases such as muscular dystrophy where nuclei occupy a central rather than peripheral location. Moreover, rearward nuclear positioning is typical of migratory cells. Active nuclear movement in most cases involves coupling of cytoskeletal components with the nucleus by a group of transmembrane proteins in the nuclear envelope called the LINC (linker of nucleoskeleton and cytoskeleton) complex. It is composed of the inner nuclear membrane SUN (Sad1p, UNC-84) proteins associated with nuclear lamins and the outer nuclear membrane KASH (Klarsicht, ANC-1, Syne Homology) proteins, which interact with the cytoskeleton. In my thesis, the murine fibroblast cell line NIH3T3 was used as a model system to study nuclear positioning in states of active movement and static homeostatic positioning. Nuclear positioning and centrosome reorientation are hallmarks of cell polarity in migrating fibroblasts. The Gundersen lab has established that the nucleus moves rearward to orient the centrosome in serum starved fibroblast monolayers stimulated by the serum-derived factor lysophosphatidic acid (LPA). LPA stimulates the GTPase Cdc42, which in turn activates the Cdc42 effector MRCK to phosphorylate myosin II and activate actin retrograde flow to move the nucleus to the rear. A second Cdc42 effector, Par6 functions with Par3 and dynein to maintain the centrosome in the cell centroid. The nucleus is moved rearward by the attachment of retrograde dorsal actin cables to the nucleus through transmembrane actin-associated nuclear (TAN) lines. TAN lines are composed linear arrays of the LINC complex proteins nesprin-2G (N2G) and SUN2 and dorsal actin cables. Disrupting TAN lines components blocks nuclear movement and efficient cell migration. Interestingly, TAN lines are analogous to other membrane adhesions, such as focal adhesions, in that they are transmembrane structures linked to the actin cytoskeleton and transmit force. Given the large number of proteins composing structures such as focal adhesions, we predicted there would be additional components in TAN lines necessary for their formation and function. Thus, I set out to identify and study cytoplasmic factors required for TAN line formation and/or function during active nuclear positioning in fibroblast. A collaborator detected N2G as a hit in a yeast two-hybrid screen for FHOD1 interactors. FHOD1 is an actin regulator and belongs to the formin family. Like other formin family members, it has an FH2 actin binding domain, an FH1 domain and DID and DAD domains that interact to autoinhibit FHOD1. Unlike other formins, FHOD1 is not activated by GTPase binding and contains a second actin binding domain (ABS domain), giving it actin bundling activity. We show that spectrin repeats (SRs) 10-13 of N2G and the N-terminus of FHOD1 interacts with each other directly by biochemical assays with purified proteins. SiRNA against FHOD1 and overexpression of either FHOD1 or N2G interacting domains prevented LPA-stimulated nuclear movement in wounded monolayers of NIH3T3 fibroblasts, suggesting that the interaction between FHOD1 and N2G is required for nuclear movement and centrosome reorientation. FHOD1 was required for TAN line formation, but was dispensable for the formation of dorsal actin cables and retrograde actin flow. By re-expressing an artificial construct containing the N2G-binding domain of FHOD1 and the actin-binding domain of α–actinin in FHOD1 depleted cells, we show that the FHOD1 ABS domain provides N2G with an additional contact to actin filaments required for nuclear movement. This study thus identifies FHOD1 as a new TAN line component and suggests that the interaction of FHOD1 with N2G may reinforce TAN lines so that they can resist the force necessary to move the nucleus. The above study identifies a new component in a pathway that actively moves the nucleus. We have far less knowledge about the mechanism that maintains the nucleus in position when it is not moving. For example, it is unknown whether the static nuclear positioning is an active process or simply an inactivation of mechanisms that actively move nuclei. To answer this question, I developed a novel method to artificially displace the nucleus in adherent cells by centrifugation and used this system to identify active mechanisms of homeostatic nuclear positioning. By subjecting wounded monolayers of starved NIH3T3 fibroblast on coverslips to centrifugal force perpendicular to the wound, I find that nuclei are displaced towards the direction of centrifugal force, so that on one wound edge, the nuclei are in the cell rear while on the other, in the cell front. After returning centrifuged cells to the incubator, I used fixed and live cell recordings to show that the displaced nuclei actively re-center within one hour, although nuclei moving rearward did so faster than those moving forward. Treating centrifuged cells with cytoskeletal drugs, revealed an actin/myosin II-dependent rearward recentration and a microtubule (MT)/dynein-dependent forward recentration. I knocked down LINC complex components to test their involvement in these movements. N2G was required for both rearward and forward movement while SUN1 and SUN2 were required for forward and rearward movement, respectively. Overexpression of different N2G constructs in N2G-depleted cells showed that different regions of N2G were necessary for each direction of movement: N-terminal constructs rescued rearward nuclear recentration whereas C-terminal constructs rescued forward recentration. Based on the minimal N2G construct that rescued forward (MT dependent) nuclear recentration, I identified a dynein and dynactin site in the C terminus of N2G. To test whether the homeostatic nuclear positioning mechanisms were active in uncentrifuged cells, I depleted cells of nesprin-2 and then re-expressed nesprin-2 constructs capable of interacting with actin, MTs or both cytoskeletal elements. Nuclei in nesprin-2-depleted cells were no longer maintained at the cell centroid and only re-expression of a construct that contained sites for interaction with both actin and MTs rescued this defect. Thus, both actin- and MT- interaction domains of N2G are required for homeostatic nuclear positioning. To test whether the actin and MT activities of N2G were important for cell migration, I depleted NIH3T3 fibroblasts of nesprin-2 and re-expressed N2G constructs capable of interaction with actin, MTs or both and tested these cells in single and collective cell migration assays. I found that only the MT-dependent activity of N2G is required for the directionality of single cell migration while both N- and C- terminal (actin- and MT- dependent) N2G are required for the velocity of collective cell migration. These results show that different cytoskeletal linkages are used in different modes of cell migration. My thesis studies identify the first cytoplasmic factor required for TAN lines structure, establish a novel method to artificially displace the nucleus in adherent cells, and reveal different mechanisms of LINC complex coupling cytoskeletons during active and homeostatic nuclear positioning, as well as specific cytoskeleton-dependent contributions of nuclear envelope protein N2G during cell migration

Symmetries and conservation laws of a nonlinear sigma model with gravitino

We show that the action functional of the nonlinear sigma model with gravitino considered in a previous article [18] is invariant under rescaled conformal transformations, super Weyl transformations and diffeomorphisms. We give a careful geometric explanation how a variation of the metric leads to the corresponding variation of the spinors. In particular cases and despite using only commutative variables, the functional possesses a degenerate super symmetry. The corresponding conservation laws lead to a geometric interpretation of the energy-momentum tensor and supercurrent as holomorphic sections of appropriate bundles.Comment: 27 page

Regularity of Solutions of the Nonlinear Sigma Model with Gravitino

We propose a geometric setup to study analytic aspects of a variant of the super symmetric two-dimensional nonlinear sigma model. This functional extends the functional of Dirac-harmonic maps by gravitino fields. The system of Euler--Lagrange equations of the two-dimensional nonlinear sigma model with gravitino is calculated explicitly. The gravitino terms pose additional analytic difficulties to show smoothness of its weak solutions which are overcome using Rivi\`ere's regularity theory and Riesz potential theory.Comment: 24 pages. This is a revised version, with some typos corrected. To appear in Commun. Math. Phy

Geometric analysis of the Yang-Mills-Higgs-Dirac model

The harmonic sections of the Kaluza-Klein model can be seen as a variant of harmonic maps with additional gauge symmetry. Geometrically, they are realized as sections of a fiber bundle associated to a principal bundle with a connection. In this paper, we investigate geometric and analytic aspects of a model that combines the Kaluza-Klein model with the Yang-Mills action and a Dirac action for twisted spinors. In dimension two we show that weak solutions of the Euler-Lagrange system are smooth. For a sequence of approximate solutions on surfaces with uniformly bounded energies we obtain compactness modulo bubbles, namely, energy identities and the no-neck property hold.Comment: 31 page

Regional Geological Survey of Hanggai, Xianxia and Chuancun, Zhejiang Province in China

This Open Access book introduces readers to the regional geology of Hanggai, Xianxia and Chuancun, the area between China's northern Zhejiang Province and southern Anhui Province and explores the strata, magmatic rocks and tectonic structures in 1:50,000 scale geological maps. Based on studies of multiple stratigraphic divisions, the standard stratigraphic section of the upper Ordovician Hirnantian in the lower Yangtze region is established, revealing for the first time numerous “Burgess Shale-type” sponge fossils in Hirnantian strata and identifying 10 grapholite fossil belts and various fossil categories, including chitin, trilobites, gastropods, brachiopods, and cephalopods. Moreover, the book identifies for the first time Late Ordovician volcanic events in northern Zhejiang province. The work represents a major contribution to research on Paleozoic strata in the Lower Yangtze region, and sheds new light on understanding the Hirnantian glacial event and biological extinction event in South China by providing a high-precision time scale. In addition, the book opens an important avenue for future research on sponge evolution after the Cambrian life explosion. As such, it offers a unique and valuable asset for researchers and graduate students alike

Identification and characterization of the critical genes encoding Cd-induced enhancement of SOD isozymes activities in Zhe-Maidong (Ophiopogon japonicus)

Superoxide dismutase (SOD) protects plants from abiotic stress-induced reactive oxygen species (ROS) damage. Here, the effects of cadmium (Cd) exposure on ROS accumulation and SOD isozymes, as well as the identification of significant SOD isozyme genes, were investigated under different Cd stress treatments to Zhe-Maidong (Ophiopogon japonicus). The exposure to Cd stress resulted in a notable elevation in the SOD activity in roots. Cu/ZnSODa and Cu/ZnSODb were the most critical SOD isozymes in response to Cd stress, as indicated by the detection results for SOD isozymes. A total of 22 OjSOD genes were identified and classified into three subgroups, including 10 OjCu/ZnSODs, 6 OjMnSODs, and 6 OjFeSODs, based on the analysis of conserved motif and phylogenetic tree. Cu/ZnSOD-15, Cu/ZnSOD-18, Cu/ZnSOD-20, and Cu/ZnSOD-22 were the main genes that control the increase in SOD activity under Cd stress, as revealed via quantitative PCR and transcriptome analysis. Additionally, under various heavy metal stress (Cu2+, Fe2+, Zn2+, Mn2+), Cu/ZnSOD-15, Cu/ZnSOD-18, and Cu/ZnSOD-22 gene expression were significantly upregulated, indicating that these three genes play a critical part in resisting heavy metal stress. The molecular docking experiments performed on the interaction between oxygen ion (O2•−) and OjSOD protein have revealed that the critical amino acid residues involved in the binding of Cu/ZnSOD-22 to the substrate were Pro135, Ile136, Ile140, and Arg144. Our findings provide a solid foundation for additional functional investigations on the OjSOD genes, as well as suggestions for improving genetic breeding and agricultural management strategies to increase Cd resistance in O. japonicus

Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes

Single nucleotide polymorphisms at the TRAF1/C5 locus are associated with rheumatoid arthritis in a Han Chinese population

<p>Abstract</p> <p>Background</p> <p>Genetic variants in <it>TRAF1C5 </it>and <it>PTPN22 </it>genes have been shown to be significantly associated with arthritis rheumatoid in Caucasian populations. This study investigated the association between single nucleotide polymorphisms (SNPs) in <it>TRAF1/C5 </it>and <it>PTPN22 </it>genes and rheumatoid arthritis (RA) in a Han Chinese population. We genotyped SNPs rs3761847 and rs7021206 at the <it>TRAF1/C5 </it>locus and rs2476601 SNP in the <it>PTPN22 </it>gene in a Han Chinese cohort composed of 576 patients with RA and 689 controls. The concentrations of anti-cyclic citrullinated peptide antibodies (CCP) and rheumatoid factor (RF) were determined for all affected patients. The difference between the cases and the controls was compared using <it>χ</it>²analysis.</p> <p>Results</p> <p>Significant differences in SNPs rs3761847 and rs7021206 at <it>TRAF1/C5 </it>were observed between the case and control groups in this cohort; the allelic p-value was 0.0018 with an odds ratio of 1.28 for rs3761847 and 0.005 with an odds ratio of 1.27 for rs7021206. This significant association between rs3761847 and RA was independent of the concentrations of anti-CCP and RF. No polymorphism of rs2476601 was observed in this cohort.</p> <p>Conclusions</p> <p>We first demonstrated that genetic variants at the <it>TRAF1/C5 </it>locus are significantly associated with RA in Han Chinese, suggesting that <it>TRAF1/C5 </it>may play a role in the development of RA in this population, which expands the pathogenesis role of <it>TRAF1/C5 </it>in a different ethnicity.</p

Efficacy and safety of low-dose IL-2 in the treatment of systemic lupus erythematosus: A randomised, double-blind, placebo-controlled trial

Objectives Open-labelled clinical trials suggested that low-dose IL-2 might be effective in treatment of systemic lupus erythematosus (SLE). A double-blind and placebocontrolled trial is required to formally evaluate the safety and efficacy of low-dose IL-2 therapy. Methods A randomised, double-blind and placebocontrolled clinical trial was designed to treat 60 patients with active SLE. These patients received either IL-2 (n=30) or placebo (n=30) with standard treatment for 12 weeks, and were followed up for additional 12 weeks. IL-2 at a dose of 1 million IU or placebo was administered subcutaneously every other day for 2 weeks and followed by a 2-week break as one treatment cycle. The primary endpoint was the SLE Responder Index-4 (SRI-4) at week 12. The secondary endpoints were other clinical responses, safety and dynamics of immune cell subsets. Results At week 12, the SRI-4 response rates were 55.17% and 30.00% for IL-2 and placebo, respectively (p=0.052). At week 24, the SRI-4 response rate of IL-2 group was 65.52%, compared with 36.67% of the placebo group (p=0.027). The primary endpoint was not met at week 12. Low-dose IL-2 treatment resulted in 53.85% (7/13) complete remission in patients with lupus nephritis, compared with 16.67% (2/12) in the placebo group (p=0.036). No serious infection was observed in the IL-2 group, but two in placebo group. Besides expansion of regulatory T cells, low-dose IL-2 may also sustain cellular immunity with enhanced natural killer cells. Conclusions Low-dose IL-2 might be effective and tolerated in treatment of SThe work was supported by the National Natural Science Foundation of China (31530020,31570880,81471601,81601417 and 81701598), Peking-Tsinghua Center for Life Sciences to ZG LI, Beijing Sci-Tech Committee Z171100000417007,Clinical Medicine Plus X-Young Scholars Project of Peking University (PKU2019LCXQ013) supported by the Fundamental Research Funds for the Central Universities, Beijing Nova Program Z171100001117025, National Key Research and Development Program of China (2017YFC0909003 to DY), BellberryViertel Senior Medical Research Fellowship to DY and Beijing SL PHARM