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As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity., Competing Interests: Declaration of Interests The authors have no competing interests to declare.

CERS6 is associated with metastasis and poor prognosis in non-small cell lung cancer (NSCLC) patients through d18:1/C16:0 ceramide (C16 ceramide)-mediated cell migration, though the detailed mechanism has not been elucidated. In the present study, examinations including co-immunoprecipitation, liquid chromatography, and tandem mass spectrometry analysis were performed to identify a novel binding partner of CERS6. Among the examined candidates, LASP1 was a top-ranked binding partner, with the LIM domain possibly required for direct interaction. In accord with those findings, CERS6 and LASP1 were found to co-localize on lamellipodia in several lung cancer cell lines. Furthermore, silencing of CERS6 and/or LASP1 significantly suppressed cell migration and lamellipodia formation, whereas ectopic addition of C16 ceramide partially rescued those phenotypes. Both LASP1 and CERS6 showed co-immunoprecipitation with actin, with those interactions markedly reduced when the LASP1–CERS6 complex was abolished. Based on these findings, it is proposed that LASP1–CERS6 interaction promotes cancer cell migration.

LIM-domain proteins have been shown to be associated with heart development and diseases. Systematic studies of LIM family members at the genome-wide level, which are crucial to further understand their functions in cardiac hypertrophy, are currently lacking. Here, 70 LIM genes were identified and characterised in mice. The expression patterns of LIM genes differ greatly during cardiac development and in the case of hypertrophy. Both Crip2 and Xirp2 are differentially expressed in cardiac hypertrophy and during heart failure. In addition, the hypertrophic state of cardiomyocytes is controlled by the relative expression levels of Crip2 and Xirp2. This study provides a foundation for further understanding of the special roles of LIM proteins in mammalian cardiac development and hypertrophy.

Ajuba is a multiple LIM domain‐containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene N‐cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E‐box element within N‐cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist‐related tumour metastasis.

LIM-domain only 2 (LMO2) is a highly conserved cysteine-rich protein that is a critical regulator of hematopoietic cell development, stem cell maintenance, and is implicated in angiogenesis [1,2]. ...

The LIM domain only 1 (LMO1) gene belongs to the LMO family of genes that encodes a group of transcriptional cofactors. This group of transcriptional cofactors regulates gene transcription by acting as a key “connector” or “scaffold” in transcription complexes. All LMOs, including LMO1, are important players in the process of tumorigenesis. Unique biological features of LMO1 distinct from other LMO members, such as its tissue-specific expression patterns, interacting proteins, and transcriptional targets, have been increasingly recognized. Studies indicated that LMO1 plays a critical oncogenic role in various types of cancers, including T-cell acute lymphoblastic leukemia, neuroblastoma, gastric cancer, lung cancer, and prostate cancer. The molecular mechanisms underlying such functions of LMO1 have also been investigated, but they are currently far from being fully elucidated. Here, we focus on reviewing the current findings on the role of LMO1 in tumorigenesis, the mechanisms of its oncogenic action, and the mechanisms that drive its aberrant activation in cancers. We also briefly review its roles in the development process and non-cancer diseases. Finally, we discuss the remaining questions and future investigations required for promoting the translation of laboratory findings to clinical applications, including cancer diagnosis and treatment.

Asiatic cotton (Gossypium arboreum) cultivated as ‘desi cotton’ in India, is renowned for its climate resilience and robustness against biotic and abiotic stresses. The genome of G. arboreum is therefore, considered as a valued reserve of information for discovering novel genes or gene functions for trait improvements in the present context of cotton cultivation world-wide. In the present study, we carried out genome-wide analysis of LIM gene family in desi cotton and identified twenty LIM domain proteins (GaLIMs) which include sixteen animals CRP-like GaLIMs and four plant specific GaLIMs with presence (GaDA1) or absence (GaDAR) of UIM (Ubiquitin Interacting Motifs). Among the sixteen CRP-like GaLIMs, eleven had two conventional LIM domains while, five had single LIM domain which was not reported in LIM gene family of the plant species studied, except in Brassica rapa. Phylogenetic analysis of these twenty GaLIM proteins in comparison with LIMs of Arabidopsis, chickpea and poplar categorized them into distinct αLIM1, βLIM1, γLIM2, δLIM2 groups in CRP-like LIMs, and GaDA1 and GaDAR in plant specific LIMs group. Domain analysis had revealed consensus [(C-X2-C-X17-H-X2-C)-X2-(C-X2-C-X17-C-X2-H)] and [(C-X2-C-X17-H-X2-C)-X2-(C-X4-C-X15-C-X2-H)] being conserved as first and/or second LIM domains of animal CRP-like GaLIMs, respectively. Interestingly, single LIM domain containing GaLIM15 was found to contain unique consensus with longer inter-zinc-motif spacer but shorter second zinc finger motif. All twenty GaLIMs showed variable spatio-temporal expression patterns and accordingly further categorized into distinct groups of αLIM1, βLIM1, γLIM2 δLIM2 and plant specific LIM (DA1/DAR). For the first time, response of GaDA1/DAR under the influence of biotic and abiotic stresses were studied in cotton, involving treatments with phytohormones (Jasmonic acid and Abscisic acid), salt (NaCl) and wilt causing pathogen (Fusarium oxysporum). Expressions patterns of GaDA1/DAR showed variable response and identified GaDA2 as a probable candidate gene for stress tolerance in G. arboreum.

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin’s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state.

Nail-patella syndrome (NPS) is an autosomal-dominant genetic disorder characterized by malformed fingernails and hypoplastic or completely absent kneecaps. In addition, ~40% of NPS patients develop a nephropathy caused by malfunctioning podocytes. Mutations in the gene LMX1B which encodes a transcription factor of the LIM-homeodomain family are known to be the cause of NPS. It is still not clear what pathogenic mechanism underlies NPS. Notably, only homozygous Lmx1b knock-out mice develop symptoms resembling those in human NPS patients. We therefore hypothesized that human patients suffer from missense mutations which confer either a loss-of-function or a dominant-negative effect. To identify possible molecular pathogenic mechanisms of NPS, Lmx1b knock-in mice were established with point mutations either in the first or the second LIM domain such as those described in patients. Heterozygous Lmx1b knock-in mice showed no NPS phenotype whereas homozygous Lmx1b knock-in mice displayed a reduced number of filtration slits per ��m basement membrane and premature ossification of the skeleton, similar to that of the conventional Lmx1b knock-out animals. The presence of the mutated Lmx1b mRNA was confirmed but the protein was not detectable in podocytes. Detailed analyses of mutated LMX1B showed a reduced half-life of H54L and C95F LMX1B mutant proteins in comparison to wild-type LMX1B. Another mutant LMX1B protein with a mutation in the homeodomain, V242D, showed a prolonged half-life compared to the wild-type protein. In addition, the pathway of LMX1B protein degradation was initially demonstrated to be accomplished by the proteasome system. Finally, in preliminary experiments, the effect on proteinuria after application of the proteasomal inhibitor bortezomib, PS-341, was examined in compound heterozygous Lmx1b C95F/loxP. First results revealed a reduced proteinuria in compound heterozygous Lmx1b C95F/loxP after podocyte-specific Lmx1b knock-out. Due to protein instability, the knock-in mutations H54L and C95F lead to a loss of function in Lmx1b knock-in mice. As previously described by Cross et al. (2014), the mutation V242D within the homeodomain leads to a dominant-negative effect which can be explained by an increased stability of the mutant protein. This suggests that different NPS mutations can lead to different pathogenic mechanisms requiring distinct therapeutical approaches., Nagel-Patella-Syndrom (NPS) ist eine autosomal dominant vererbbare Krankheit und wird durch fehlgeformte und hypoplastische oder vollst��ndig fehlende Kniescheiben charakterisiert. Zus��tzlich entwickeln ~40% der NPS Patienten eine Nephropathie, die durch Versagen der Podozytenstruktur verursacht wird. Mutationen im Gen LMX1B, welches f��r den Transkriptionsfaktor der LIM-Hom��odom��nen Familie codiert, verursachen NPS. Es ist bislang nicht gekl��rt, welcher Pathomechanismus NPS unterliegt. Bemerkenswerterweise entwickeln nur homozygote Lmx1b knock-out M��use einen Ph��notyp, welcher der Symptomatik von NPS Patienten ��hnelt. Daher stellt sich die Frage, ob die missense Mutationen entweder eine loss-of-function oder einen dominant-negativen Effekt hervorrufen. Diese Frage sollte mittels der Charakterisierung von Lmx1b knock-in Mauslinien, Lmx1b (H54L) und Lmx1b (C95F), welche Patienten basierte Mutationen aufweisen, beantwortet werden. Heterozygote Lmx1b knock-in M��use zeigten keinen NPS Ph��ntyp, w��hrend homozygote Lmx1b knock-in M��use eine reduzierte Anzahl an Filtrationsschlitzen pro ��m glomerul��rer Basalmembran aufzeigten und eine vorzeitige Ossifikation des Skeletts aufwiesen. Die mutierte Lmx1b mRNA wurde best��tigt, jedoch war das Lmx1b Protein in Podozyten nicht detektierbar. Detaillierte Analysen des mutierten Proteins zeigten eine verk��rzte Halbwertszeit von H54L und C95F mutiertem LMX1B Protein im Vergleich zum Wild-typ Protein. Ein weiteres mutiertes LMX1B Protein mit einer Mutation in der Hom��odom��ne, V242D, zeigte eine verl��ngerte Halbwertszeit verglichen mit dem Wild-typ Protein. Zus��tzlich wurde der Abbauweg von LMX1B ��ber das Proteasom best��tigt. Zuletzt wurde in ersten Experimenten der Effekt auf die Proteinurie nach Verabreichung des Proteasominhibitors Bortezomib (PS-341) in gemischt heterozygoten Lmx1b C95F/loxP in vivo untersucht. Erste Daten der Lmx1b C95F/loxP M��use zeigten eine reduzierte Proteinurie nach induziertem podozyten-spezifischen Lmx1b knock-out. Aufgrund der Protein Instabilit��t, f��hren die Lmx1b knock-in Mutationen H54L und C95F zu einem Funktionsverlust von LMX1B. Es wurde bereits durch Cross et al. (2014) gezeigt, dass die Mutation V242D in der Hom��odom��ne zu einem dominant-negativen Effekt f��hrt, was auch durch die erh��hte Stabilit��t des mutierten Proteins erkl��rt werden kann. Dies w��rde bedeuten, dass NPS Mutationen unterschiedliche Pathomechanismen hervorrufen, was wiederrum unterschiedliche Therapieverfahren erfordern w��rde.

AIMS An indolent T-lymphoblastic proliferation (iT-LBP) is a benign, reactive expansion of immature terminal deoxynucleotidyl transferase (TdT)-positive T cells found in extrathymic tissues. iT-LBP can be challenging to distinguish from malignant processes, specifically T-lymphoblastic lymphoma (T-LBL), given the overlapping clinical and histological features. Recently, it has been shown that LIM domain only 2 (LMO2) is overexpressed in T-LBL but not in reactive immature TdT+ T cells in the thymus. On the basis of these findings, the aim of this study was to investigate the expression of LMO2 by using immunohistochemistry and its role in differentiating iT-LBPs from T-LBLs. METHODS AND RESULTS We retrospectively identified cases of iT-LBP and T-LBL from the pathology archives of four institutions. Seven iT-LBP cases (including five new cases that have not been reported in the literature) and 13 T-LBL cases were analysed. Clinical, morphological, immunophenotypic and molecular data were analysed. Immunohistochemical staining with LMO2 was performed on all iT-LBP and T-LBL cases. A review of five new iT-LBP cases showed similar morphological, immunophenotypic and molecular features to those of previously reported cases. All iT-LBP cases were negative for LMO2 (0/7), whereas 92% of T-LBL cases (12/13) expressed LMO2; the sensitivity was 92% (confidence interval 64-100%) and the specificity was 100% (confidence interval 59-100%). CONCLUSION We confirm previously published findings that iT-LBP cases show highly overlapping morphological and immunophenotypic features with T-LBL. Importantly, LMO2 expression is a sensitive and specific marker with which to rule out iT-LBP.

Four and a half LIM domains protein 1 (FHL1), as the name suggests, contains four and a half LIM domains capable of interacting with various molecules, including structural proteins, kinases, and transcriptional machinery. FHL1 contains a zinc-finger domain and performs diverse roles in regulation of gene transcription, cytoarchitecture, cell proliferation, and signal transduction. Several studies have validated the importance of FHL1 in muscle development, myopathy, and cardiovascular diseases. Mutations in the FHL1 gene are associated with various myopathies. Recently, FHL1 was identified as a major host factor for chikungunya virus (CHIKV) infection in both humans and mice. Based on more recent findings over the last decade, FHL1 is proposed to play a dual role in cancer progression. On the one hand, FHL1 expression is suppressed in several cancer types, which correlates with increased metastatic disease and decreased survival. Moreover, FHL1 is reported to inhibit tumor cell growth and migration by associating with diverse signals, such as TGF-β and ER, and therefore considered a tumor suppressor. On the other hand, FHL1 can function as an oncogenic protein that promotes tumor progression upon phosphorylation, reflecting complex roles in cancer. This review primarily focuses on the dual role and underlying mechanisms of action of FHL1 in human cancer progression and its clinical relevance.

A male mouse exhibiting bidirectional circling behavior was identified in a Y-chromosome consomic strain known as DH-Chr YRR . The putative mutation responsible for the circling behavior was inherited in an autosomal recessive manner and was termed circ. To identify its causative gene, we performed exome sequencing; of the 34 candidates discovered, we found a novel nonsynonymous single nucleotide variation in LIM homeobox transcription factor 1 alpha (Lmx1a) (c.394G > C, p.Gly132Arg). The genetic linkage between Lmx1a and circ was confirmed in (♀BALB/cA × ♂DH-Chr YRR -circ/circ) F2 and (♀C57BL/6J × ♂DH-Chr YRR -circ/circ) F2 mice. The Lmx1a mutation led to many abnormalities that affected growth, pigmentation, reproduction, and cerebellar morphology. We showed that (♀BALB/cA × ♂DH-Chr YRR -circ/circ) F2 -circ/circ mice demonstrated significantly lower body mass than the F2 -+/? mice. Unlike the F2 -+/? mice, few (♀C57BL/6J × ♂DH-Chr YRR -circ/circ) F2 -circ/circ mice exhibited a belly spot. The circ/circ females were also invariably sterile, probably because of an underdeveloped uterus. Moreover, the circ/circ mice presented fewer cerebellar granule cells with lower density than the F2 -+/? mice. Although non-complementation between circ and the known Lmx1a mutant alleles remains unconfirmed, the coisogenic nature of circ strongly suggests that it is a novel variant of Lmx1a, previously known as dreher. Therefore, we have assigned the gene symbol Lmx1adr-circ to circ. In addition to Lmx1adr-J and Lmx1adr-kjmi , Lmx1adr-circ is the third allele that causes a missense mutation within LIM domains. Identification of missense mutations is necessary to specify the critical residues for abrogating the in vivo functions of LMX1A.

During embryonic morphogenesis, the integrity of epithelial tissues depends on the ability of cells in tissue sheets to undergo rapid changes in cell shape while preventing self-injury to junctional actin networks. LIM domain-containing repeat (LCR) proteins are recruited to sites of strained actin filaments in cultured cells [1–3], and are therefore promising candidates for mediating self-healing of actin networks, but whether they play similar roles in living organisms has not been determined. Here, we establish roles for Caenorhabditis elegans TES-1/Tes, an actin-binding LCR protein present at apical junctions, during epithelial morphogenesis. TES-1::GFP is recruited to apical junctions during embryonic elongation, when junctions are under tension; in embryos in which stochastic failure of cell elongation occurs, TES-1 is only strongly recruited to junctions in cells that successfully elongate, and recruitment is severely compromised in genetic backgrounds in which cell shape changes do not successfully occur. tes-1 mutant embryos display junctional F-actin defects, and loss of TES-1 strongly enhances tension-dependent injury of junctional actin networks in hypomorphic mutant backgrounds for CCC components, suggesting that TES-1 helps to prevent self-injury of junctional actin networks during rapid cell shape change. Consistent with such role, a fragment of TES-1 containing its LIM domains localizes to stress fiber strain sites (SFSS) in cultured vertebrate cells. Together, these data establish TES-1 as a tension-sensitive stabilizer of the junctional actin cytoskeleton during embryonic morphogenesis.

Circular RNA (circRNA) is a class of endogenous non-coding RNAs without 5′ and 3′ ends; an increasing number of studies show that circRNA is involved in skeletal muscle development. From our previous sequencing data, the circRNAome in breast muscle of two chicken lines with a distinct rate of muscle development, which included a fast muscle growing broiler (FMGB) and a slow muscle growing layer (SMGL), we found a novel differentially expressed circRNA generated by intersectin 2 (ITSN2) gene (named circITSN2). We verified that circITSN2 is a skeletal muscle-enriched circRNA that promotes chicken primary myoblast (CPM) proliferation and differentiation. Further molecular mechanism analysis of circITSN2 in chicken myogenesis was performed, and we found circITSN2 directly targeting miR-218-5p. Besides, miR-218-5p inhibits CPM proliferation and differentiation, which is contrary to circITSN2. Commonly, circRNAs act as a miRNA sponge to alleviate the inhibition of miRNAs on mRNAs. Thus, we also identified that a downstream gene LIM domain 7 (LMO7) was inhibited by miR-218-5p, while circITSN2 could block the inhibitory effect of miR-218-5p by targeting it. Functional analysis revealed that LMO7 also accelerates CPM proliferation and differentiation, which was similar to circITSN2 but contrary to miR-218-5p. Taken together, these results suggested that circITSN2 promotes chicken embryonic skeletal muscle development via relieving the inhibition of miR-218-5p on LMO7. Our findings revealed a novel circITSN2/miR-218-5p/LMO7 axis in chicken embryonic skeletal muscle development, which expands our understanding of the complex muscle development regulatory network.

Arterial hypertension is the leading risk factor for cardiovascular morbidity and mortality worldwide. However, little is known about the cellular mechanisms underlying it. In small arteries and arterioles, a chronic increase in blood pressure raises wall tension and hence stretches, namely, the medial vascular smooth muscle cells (VSMC) but also endothelial cell (EC) to cell contacts. Initially compensated by an increase in vascular tone, the continuous biomechanical strain causes a prominent change in gene expression in both cell types, frequently driving an arterial inward remodeling process that ultimately results in a reduction in lumen diameter, stiffening of the vessel wall, and fixation of blood pressure, namely, diastolic blood pressure, at the elevated level. Sensing and propagation of this supraphysiological stretch into the nucleus of VSMC and EC therefore seems to be a crucial step in the initiation and advancement of hypertension-induced arterial remodeling. Focal adhesions (FA) represent an important interface between the extracellular matrix and Lin11-Isl1-Mec3 (LIM) domain-containing proteins, which can translocate from the FA into the nucleus where they affect gene expression. The varying biomechanical cues to which vascular cells are exposed can thus be rapidly and specifically propagated to the nucleus. Zyxin was the first protein described with such mechanotransducing properties. It comprises 3 C-terminal LIM domains, a leucine-rich nuclear export signal, and N-terminal features that support its association with the actin cytoskeleton. In the cytoplasm, zyxin promotes actin assembly and organization as well as cell motility. In EC, zyxin acts as a transcription factor, whereas in VSMC, it has a less direct effect on mechanosensitive gene expression. In terms of homology and structural features, lipoma preferred partner is the nearest relative of zyxin among the LIM domain proteins. It is almost exclusively expressed by smooth muscle cells in the adult, resides like zyxin at FA but seems to affect mechanosensitive gene expression indirectly, possibly via altering cortical actin dynamics. Here, we highlight what is currently known about the role of these LIM domain proteins in mechanosensing and transduction in vascular cells.

Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.

Summary The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/454387v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@137afd1org.highwire.dtl.DTLVardef@f7c91eorg.highwire.dtl.DTLVardef@1b4f2eborg.highwire.dtl.DTLVardef@b82f7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Epithelial Protein Lost In Neoplasm (EPLIN), also known as LIMA1 (LIM Domain And Actin Binding 1), was first discovered as a protein differentially expressed in normal and cancerous cell lines. It is now known to be key to the progression and metastasis of certain solid tumours. Despite a slow pace in understanding the biological role in cells and body systems, as well as its clinical implications in the early years since its discovery, recent years have witnessed a rapid progress in understanding the mechanisms of this protein in cells, diseases and indeed the body. EPLIN has drawn more attention over the past few years with its roles expanding from cell migration and cytoskeletal dynamics, to cell cycle, gene regulation, angiogenesis/lymphangiogenesis and lipid metabolism. This concise review summarises and discusses the recent progress in understanding EPLIN in biological processes and its implications in cancer.

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains binds binds Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. We found that the N-terminal domain localizes to the basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Using a novel targeted proximity biotinylation approach with anti-GFP antibody attached to the biotin ligase BirA in the presence of GFP-Wtip-N, we identified SSX2IP as the candidate binding protein. SSX2IP, also known as Msd1 or ADIP, is a centriolar satellite protein that functions as a targeting factor for ciliary membrane proteins. Wtip physically associated with SSX2IP and the two proteins formed mixed spherical aggregates in overexpressing cells in a dose-dependent manner, in a process that resembles phase separation. These results suggest that the interaction between SSX2IP and Wtip is relevant to their functions at the centrosome and basal bodies. The described antibody targeting of biotin ligase should be applicable to other GFP-tagged proteins.

Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development, such as furrow or tube formation. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is localized to and promotes the formation of the circumferential actomyosin belt adjacent to apical junctions. We find that Lmo7-dependent apical constriction requires the RhoA-ROCK-Non-muscle myosin II (NMII) pathway. Strikingly, Lmo7 binds and recruits NMII heavy chains to apical junctions. Lmo7 overexpression altered the subcellular distribution of Wtip, a sensor of mechanical tension. Our findings suggest that Lmo7 serves as a scaffold organizing the actomyosin network to regulate contractility at apical junctions.

Skeletal muscle satellite cell growth and development is a complicated process driven by multiple genes. The PDZ and LIM domain 5 (PDLIM5) gene has been proven to function in C2C12 myoblast differentiation and is involved in the regulation of skeletal muscle development. The role of PDLIM5 in chicken skeletal muscle satellite cells, however, is unclear. In this study, in order to determine whether the PDLIM5 gene has a function in chicken skeletal muscle satellite cells, we examined the changes in proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs) after interfering and overexpressing PDLIM5 in cells. In addition, the molecular pathways of the PDLIM5 gene regulating SMSC proliferation and differentiation were analyzed by transcriptome sequencing. Our results show that PDLIM5 can promote the proliferation and differentiation of SMSCs, furthermore, through transcriptome sequencing, it can be found that the differential genes are enriched in the MAPK signaling pathway after knocking down PDLIM5. Finally, it was verified that PDLIM5 played an active role in the proliferation and differentiation of chicken SMSCs by activating the p38-MAPK signaling pathway. These results indicate that PDLIM5 may be involved in the growth and development of chicken skeletal muscle.

JRAB/MICAL-L2 is an effector protein of Rab13, a member of the Rab family of small GTPase. JRAB/MICAL-L2 consists of a calponin homology domain, a LIM domain, and a coiled-coil domain. JRAB/MICAL-L2 engages in intramolecular interaction between the N-terminal LIM domain and the C-terminal coiled-coil domain, and changes its conformation from closed to open under the effect of Rab13. Open-form JRAB/MICAL-L2 induces the formation of peripheral ruffles via an interaction between its calponin homology domain and filamin. Here, we report that the LIM domain, independent of the C-terminus, is also necessary for the function of open-form JRAB/MICAL-L2. In mechanistic terms, two zinc finger domains within the LIM domain bind the first and second molecules of actin at the minus end, potentially inhibiting the depolymerization of actin filaments (F-actin). The first zinc finger domain also contributes to the intramolecular interaction of JRAB/MICAL-L2. Moreover, the residues of the first zinc finger domain that are responsible for the intramolecular interaction are also involved in the association with F-actin. Together, our findings show that the function of open-form JRAB/MICAL-L2 mediated by the LIM domain is fine-tuned by the intramolecular interaction between the first zinc finger domain and the C-terminal domain.

Diabetes is characterized by a loss of β-cell mass, and a greater understanding of the transcriptional mechanisms governing β-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA–binding protein 3 (SSBP3) regulates the genes necessary for β-cell function, but few proteins are known to interact with this complex in β-cells. To identify additional components, here we performed SSBP3 reverse–cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse β-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for β-cell function. We observed that Rnf20 and Rnf40 depletion reduces β-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate β-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Alp/Enigma family members have a unique PDZ domain followed by zero to four LIM domains, and are essential for myofibril assembly across all species analyzed so far. Drosophila melanogaster has three Alp/Enigma family members, Zasp52, Zasp66, and Zasp67. Ortholog search and phylogenetic tree analysis suggest that Zasp genes have a common ancestor, and that Zasp66 and Zasp67 arose by duplication in insects. While Zasp66 has a conserved domain structure across orthologs, Zasp67 domains and lengths are highly variable. In flies, Zasp67 appears to be expressed only in indirect flight muscles, where it colocalizes with Zasp52 at Z-discs. We generated a CRISPR null mutant of Zasp67, which is viable but flightless. We can rescue all phenotypes by re-expressing a Zasp67 transgene at endogenous levels. Zasp67 mutants show extended and broken Z-discs in adult flies, indicating that the protein helps stabilize the highly regular myofibrils of indirect flight muscles. In contrast, a Zasp66 CRISPR null mutant has limited viability, but only mild indirect flight muscle defects illustrating the diverging evolutionary paths these two paralogous genes have taken since they arose by duplication.

Smallish (Smash), the Drosophila homologue of human LIM domain only 7 (LMO7), is a key regulator of Drosophila embryogenesis associated with planner cell polarity and actomyosin contractility at the zonula adherence. Although smash mRNA is expressed in several tissues during Drosophila development, only Smash function at the adherence junction in the embryonic epithelial cells has been reported. We herein demonstrated that the knockdown of smash in eye imaginal discs induced morphological aberrations in adult compound eyes that were associated with increased apoptosis. Furthermore, immunohistochemical analyses revealed that Smash localized to the nucleus in several tissues, including eye imaginal discs. The knockdown of smash in eye imaginal discs down-regulated the expression of the ote and bocks genes as well as the Drosophila homologue of the emerin gene, which is a target of LMO7. Collectively, these results indicate that Smash functions in proper Drosophila eye development mediated by the regulation of ote and bocks gene expression.

Background The Notch signaling pathway governs the specification of different cell types in flies, nematodes and vertebrates alike. Principal components of the pathway that activate Notch target genes are highly conserved throughout the animal kingdom. Despite the impact on development and disease, repression mechanisms are less well studied. Repressors are known from arthropods and vertebrates that differ strikingly by mode of action: whereas Drosophila Hairless assembles repressor complexes with CSL transcription factors, competition between activator and repressors occurs in vertebrates (for example SHARP/MINT and KyoT2). This divergence raises questions on the evolution: Are there common ancestors throughout the animal kingdom? Results Available genome databases representing all animal clades were searched for homologues of Hairless, SHARP and KyoT2. The most distant species with convincing Hairless orthologs belong to Myriapoda, indicating its emergence after the Mandibulata-Chelicarata radiation about 500 million years ago. SHARP shares motifs with SPEN and SPENITO proteins, present throughout the animal kingdom. The CSL interacting domain of SHARP, however, is specific to vertebrates separated by roughly 600 million years of evolution. KyoT2 bears a C-terminal CSL interaction domain (CID), present only in placental mammals but highly diverged already in marsupials, suggesting introduction roughly 100 million years ago. Based on the LIM-domains that characterize KyoT2, homologues can be found in Drosophila melanogaster (Limpet) and Hydra vulgaris (Prickle 3 like). These lack the CID of KyoT2, however, contain a PET and additional LIM domains. Conservation of intron/exon boundaries underscores the phylogenetic relationship between KyoT2, Limpet and Prickle. Most strikingly, Limpet and Prickle proteins carry a tetra-peptide motif resembling that of several CSL interactors. Overall, KyoT2 may have evolved from prickle and Limpet to a Notch repressor in mammals. Conclusions Notch repressors appear to be specific to either chordates or arthropods. Orthologues of experimentally validated repressors were not found outside the phylogenetic group they have been originally identified. However, the data provide a hypothesis on the evolution of mammalian KyoT2 from Prickle like ancestors. The finding of a potential CSL interacting domain in Prickle homologues points to a novel, very ancestral CSL interactor present in the entire animal kingdom. Electronic supplementary material The online version of this article (10.1186/s41065-019-0081-0) contains supplementary material, which is available to authorized users.

Certain animal and plant pathogenic bacteria have developed virulence factors including effector proteins that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto) secretes a large repertoire of effectors via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that Pto causes sepsis in mice. Surprisingly, the effector HopQ1 disrupted animal phagocytosis by inhibiting actin rearrangement via direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide novel insight into animal host-plant pathogen interactions. In addition, the current study firstly demonstrates that certain plant pathogenic bacteria such as Pto evade phagocytosis by animal cells due to cross-kingdom suppression of host immunity.

Drosophila melanogasterhas been used as a model organism for study on development and pathophysiology of the heart. LIM domain proteins act as adaptors or scaffolds to promote the assembly of multimeric protein complexes. We found a total of 75 proteins encoded by 36 genes have LIM domain inDrosophila melanogasterby the tools of SMART, FLY-FISH, and FlyExpress, and around 41.7% proteins with LIM domain locate in lymph glands, muscles system, and circulatory system. Furthermore, we summarized functions of different LIM domain proteins in the development and physiology of fly heart and hematopoietic systems. It would be attractive to determine whether it exists a probable “LIM code” for the cycle of different cell fates in cardiac and hematopoietic tissues. Next, we aspired to propose a new research direction that the LIM domain proteins may play an important role in fly cardiac and hematopoietic morphogenesis.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00853-z.

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes.

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a ‘closed’ conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis., Cilia, tiny outgrowths of cells, are essential for life. Here, the author’s describe the scaffold protein TRIP6, which promotes the assembly of ciliary proteins required for ciliogenesis, and show that its absence results in hydrocephalus.