Overexpression of the limk1 Gene in Drosophila melanogaster Can Lead to Suppression of Courtship Memory in Males.

Courtship suppression is a behavioral adaptation of the fruit fly. When majority of the females in a fly population are fertilized and non-receptive for mating, a male, after a series of failed attempts, decreases its courtship activity towards all females, saving its energy and reproductive resources. The time of courtship decrease depends on both duration of unsuccessful courtship and genetically determined features of the male nervous system. Thereby, courtship suppression paradigm can be used for studying molecular mechanisms of learning and memory. p-Cofilin, a component of the actin remodeling signaling cascade and product of LIM-kinase 1 (LIMK1), regulates Drosophila melanogaster forgetting in olfactory learning paradigm. Previously, we have shown that limk1 suppression in the specific types of nervous cells differently affects fly courtship memory. Here, we used Gal4 > UAS system to induce limk1 overexpression in the same types of neurons. limk1 activation in the mushroom body, glia, and fruitless neurons decreased learning index compared to the control strain or the strain with limk1 knockdown. In cholinergic and dopaminergic/serotoninergic neurons, both overexpression and knockdown of limk1 impaired Drosophila short-term memory. Thus, proper balance of the limk1 activity is crucial for normal cognitive activity of the fruit fly.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization.

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

Autoregulation of the LIM kinases by their PDZ domain.

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the 'x-Φ-G-Φ' motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

LIMK1 m6A-RNA methylation recognized by YTHDC2 induces 5-FU chemoresistance in colorectal cancer via endoplasmic reticulum stress and stress granule formation.

LIM kinase 1 (LIMK1) is a member of the LIMK family that has been considered to be involved in chemoresistance in various tumors, and N6-methyladenosine (m6A) is the most abundant nucleotide modification on mRNA. However, whether elevated expression of LIMK1 leads to chemoresistance due to m6A modification remains to be further studied. The findings of our study indicate that high LIMK1 expression in colorectal cancer (CRC) cells promotes cell proliferation and increases resistance to 5-fluorouracil (5-FU). Moreover, downregulation of YTH domain-containing 2 (YTHDC2), an m6A "reader", in CRC cells resulted in decreased recognition and binding to the m6A site "GGACA" in LIMK1 mRNA, thereby increasing LIMK1 mRNA stability and expression. Furthermore, the overexpression of LIMK1 facilitated eIF2α phosphorylation, which induced endoplasmic reticulum (ER) stress and promoted stress granule (SG) formation, ultimately leading to 5-FU resistance. This study evaluated the specificity of the YTHDC2/LIMK1/eIF2α signalling axis and the efficacy of related drugs in modulating 5-FU sensitivity in CRC.

Dorsal visual stream and LIMK1: hemideletion, haplotype, and enduring effects in children with Williams syndrome.

BACKGROUND: Williams syndrome (WS), a rare neurodevelopmental disorder caused by hemizygous deletion of ~ 25 genes from chromosomal band 7q11.23, affords an exceptional opportunity to study associations between a well-delineated genetic abnormality and a well-characterized neurobehavioral profile. Clinically, WS is typified by increased social drive (often termed "hypersociability") and severe visuospatial construction deficits. Previous studies have linked visuospatial problems in WS with alterations in the dorsal visual processing stream. We investigated the impacts of hemideletion and haplotype variation of LIMK1, a gene hemideleted in WS and linked to neuronal maturation and migration, on the structure and function of the dorsal stream, specifically the intraparietal sulcus (IPS), a region known to be altered in adults with WS. METHODS: We tested for IPS structural and functional changes using longitudinal MRI in a developing cohort of children with WS (76 visits from 33 participants, compared to 280 visits from 94 typically developing age- and sex-matched participants) over the age range of 5-22. We also performed MRI studies of 12 individuals with rare, shorter hemideletions at 7q11.23, all of which included LIMK1. Finally, we tested for effects of LIMK1 variation on IPS structure and imputed LIMK1 expression in two independent cohorts of healthy individuals from the general population. RESULTS: IPS structural (p < 10-4 FDR corrected) and functional (p < .05 FDR corrected) anomalies previously reported in adults were confirmed in children with WS, and, consistent with an enduring genetic mechanism, were stable from early childhood into adulthood. In the short hemideletion cohort, IPS deficits similar to those in WS were found, although effect sizes were smaller than those found in WS for both structural and functional findings. Finally, in each of the two general population cohorts stratified by LIMK1 haplotype, IPS gray matter volume (pdiscovery < 0.05 SVC, preplication = 0.0015) and imputed LIMK1 expression (pdiscovery = 10-15, preplication = 10-23) varied according to LIMK1 haplotype. CONCLUSIONS: This work offers insight into neurobiological and genetic mechanisms responsible for the WS phenotype and also more generally provides a striking example of the mechanisms by which genetic variation, acting by means of molecular effects on a neural intermediary, can influence human cognition and, in some cases, lead to neurocognitive disorders.

LIM Kinases: From Molecular to Pathological Features.

LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of the LIM kinase family harbouring two LIM domains at their N-terminus and a kinase domain at their C-terminus [...].

LIMK2: A Multifaceted kinase with pleiotropic roles in human physiology and pathologies.

LIMK2, a serine-specific kinase, was discovered as an actin dynamics regulating kinase. Emerging studies have shown its pivotal role in numerous human malignancies and neurodevelopmental disorder. Inducible knockdown of LIMK2 fully reverses tumorigenesis, underscoring its potential as a clinical target. However, the molecular mechanisms leading to its upregulation and its deregulated activity in various diseases largely remain unknown. Similarly, LIMK2's peptide substrate specificity has not been analyzed. This is particularly important for LIMK2, a kinase almost three decades old, as only a handful of its substrates are known to date. As a result, most of LIMK2's physiological and pathological roles have been assigned to its regulation of actin dynamics via cofilin. This review focuses on LIMK2's unique catalytic mechanism, substrate specificity and its upstream regulators at transcriptional, post-transcriptional and post-translational stages. Moreover, emerging studies have unveiled a few tumor suppressors and oncogenes as LIMK2's direct substrates, which in turn have uncovered novel molecular mechanisms by which it plays pleiotropic roles in human physiology and pathologies independent of actin dynamics.

LIMK2 promotes melanoma tumor growth and metastasis through G3BP1-ESM1 pathway-mediated apoptosis inhibition.

Melanoma is the leading cause of skin cancer-related deaths, and current melanoma therapies, including targeted therapies and immunotherapies, benefit only a subset of metastatic melanoma patients due to either intrinsic or acquired resistance. LIM domain kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIMK2 is overexpressed in melanoma, and its genetic or pharmacological inhibition impairs melanoma tumor growth and metastasis in both cell culture and mice. To determine the mechanism by which LIMK2 promotes melanoma tumor growth and metastatic progression, we performed a phosphoproteomics analysis and identified G3BP1 as a key LIMK2 target, which mirrored the effects of LIMK2 inhibition when inhibited. To further determine the role of G3BP1 downstream of LIMK2, we knocked down the expression of G3BP1, performed RNA-seq analysis, and identified ESM1 as a downstream target of G3BP1. G3BP1 was required for ESM1 mRNA stability, and ESM1 ectopic expression rescued LIMK2 or G3BP1 inhibition-induced suppression of melanoma growth and metastatic attributes. These results collectively identify the LIMK2→G3BP1→ESM1 pathway as a facilitator of melanoma tumor growth and metastasis and document that LIMK2 is a therapeutically tractable target for melanoma therapy.

Exosomal miR-374c-5p derived from mesenchymal stem cells suppresses epithelial-mesenchymal transition of hepatocellular carcinoma via the LIMK1-Wnt/ß-catenin axis.

Metastasis is a leading cause to treatment failure in hepatocellular carcinoma (HCC) patients. Exosomes act as pivotal mediators in communication between different cells and exert effects on recipient cells by delivering bioactive cargoes, such as microRNAs (miRNAs). MiRNAs function in multiple steps of HCC development, including metastasis. MiR-374c-5p was previously identified as a tumor suppressor in some malignancies, while the current knowledge of its role in HCC metastasis is still limited. Herein, miR-374c-5p was found to be downregulated in HCC cell lines and clinical samples, and positively related with favorable prognosis in HCC patients. MiR-374c-5p transferred by exosomes derived from bone marrow mesenchymal stem cell (BMSC) suppressed migration, invasion and proliferation of HCC cells. LIMK1 was verified as downstream target gene of miR-374c-5p. Knockdown of LIMK1 reduced invasion, migration and proliferation of HCC cells, whereas overexpression functioned oppositely. The miR-374c-5p/LIMK1 axis suppressed epithelial-mesenchymal transition (EMT) by inactivating Wnt/ß-catenin pathway. In addition, miR-374c-5p was downregulated and LIMK1 upregulated in TGF-ß1 induced EMT. This EMT model could be reversed by LIMK1 silencing or miR-374c-5p overexpression. These results suggest that exo-miR-374c-5p suppresses EMT via targeting LIMK1-Wnt/ß-catenin axis and the axis is involved in TGF-ß1 induced metastasis of HCC, thereby identifying miR-374c-5p as a potential target for HCC treatment.

Diallyl disulfide downregulating RhoGDI2 induces differentiation and inhibit invasion via the Rac1/Pak1/LIMK1 pathway in human leukemia HL-60 cells.

Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.

Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes.

LIMKs are important regulators of actin and microtubule dynamics, and they play essential roles in many cellular processes. Deregulation of LIMKs has been linked to the development of diverse diseases, including cancers and cognitive disabilities, but well-characterized inhibitors known as chemical probes are still lacking. Here, we report the characterization of three highly selective LIMK1/2 inhibitors covering all canonical binding modes (type I/II/III) and the structure-based design of the type II/III inhibitors. Characterization of these chemical probes revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed excellent selectivity in a comprehensive scanMAX kinase selectivity panel. Phosphoproteomics revealed remarkable differences between type I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models of fragile X-chromosome, 15 showed promising activity, suggesting the potential application of allosteric LIMK inhibitors treating this orphan disease.

A genome-wide association study for rheumatoid arthritis replicates previous HLA and non-HLA associations in a cohort from South Africa.

The complex pathogenesis of rheumatoid arthritis (RA) is not fully understood, with few studies exploring the genomic contribution to RA in patients from Africa. We report a genome-wide association study (GWAS) of South-Eastern Bantu-Speaking South Africans (SEBSSAs) with seropositive RA (n = 531) and population controls (n = 2653). Association testing was performed using PLINK (logistic regression assuming an additive model) with sex, age, smoking and the first three principal components as covariates. The strong association with the Human Leukocyte Antigen (HLA) region, indexed by rs602457 (near HLA-DRB1), was replicated. An additional independent signal in the HLA region represented by the lead SNP rs2523593 (near the HLA-B gene; Conditional P-value = 6.4 × 10-10) was detected. Although none of the non-HLA signals reached genome-wide significance (P < 5 × 10-8), 17 genomic regions showed suggestive association (P < 5 × 10-6). The GWAS replicated two known non-HLA associations with MMEL1 (rs2843401) and ANKRD55 (rs7731626) at a threshold of P < 5 × 10-3 providing, for the first time, evidence for replication of non-HLA signals for RA in sub-Saharan African populations. Meta-analysis with summary statistics from an African-American cohort (CLEAR study) replicated three additional non-HLA signals (rs11571302, rs2558210 and rs2422345 around KRT18P39-NPM1P33, CTLA4-ICOS and AL645568.1, respectively). Analysis based on genomic regions (200 kb windows) further replicated previously reported non-HLA signals around PADI4, CD28 and LIMK1. Although allele frequencies were overall strongly correlated between the SEBSSA and the CLEAR cohort, we observed some differences in effect size estimates for associated loci. The study highlights the need for conducting larger association studies across diverse African populations to inform precision medicine-based approaches for RA in Africa.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

OBJECTIVE: To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. METHOD: Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. RESULTS: Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1ß and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1ß and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). CONCLUSION: In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

LIMK2 Is a Novel Prognostic Biomarker and Correlates With Tumor Immune Cell Infiltration in Lung Squamous Cell Carcinoma.

Previous research found that LIM domain kinase 2 (LIMK2) expression correlated with a poor prognosis in many cancers. However, its role in lung squamous cell carcinoma (LUSC) has not yet been clarified. Our study aimed to clarify the role of LIMK2 in LUSC prognosis prediction and explore the relationship between LIMK2 and immune infiltration in LUSC. In this study, we first analyzed the expression level and prognostic value of LIMK2 across cancers. Subsequently, we explored the association of LIMK2 expression with immune infiltrating cells and immune checkpoints. our study found that LIMK2 was highly expressed and positively associated with the overall survival of LUSC. Moreover, our study further indicated that LIMK2 expression was significantly negatively correlated with immune cell infiltration and immune checkpoints in LUSC. Finally, we confirmed upstream regulatory noncoding RNAs (ncRNAs) of LIMK2, and the PVT1 and DHRS4-AS1/miR-423-5p/LIMK2 regulatory axes were successfully constructed in LUSC. Put together, LIMK2 is a novel prognostic biomarker and correlates with tumor immune cell infiltration in LUSC, and the expression of LIMK2 is regulated by the PVT1 and DHRS4-AS1/miR-423-5p axes.

The abnormal phosphorylation of the Rac1, Lim-kinase 1, and Cofilin proteins in the pathogenesis of Hirschsprung's disease.

Rac1 can affect the migration of neural crest cells by regulating the polymerization of actin and the membrane formation process. But the role of the Rac1 signaling pathway in the pathogenesis of Hirschsprung's disease (HSCR) remains unclear. In order to investigate the mechanism of the abnormal protein phosphorylation of Rac1, Lim-kinase 1 (Limk1) and Cofilin involved in the pathogenesis of HSCR. The protein phosphorylation levels of these proteins were detected by Western blot in 30 samples of HSCR narrow segment, 30 samples of transitional segment tissues, and 14 samples of normal intestinal tissues. Subsequently, in the SH-SY5Y human neuroblastoma cell line, a Rac1, Limk1, and Cofilin inhibitor group, a Rac1 overexpression group (PDGF-BB group), a Rac1 overexpression group + a Limk1 inhibitor group (P-B group), a Rac1 overexpression group + a Cofilin inhibitor group (P-C group) were established. The results showed that the expressions of p-Rac1, p-Limk1, and p-Cofilin in HSCR narrow segment and transitional segment were lower than those in normal intestine (p < 0.05). The expression levels of p-Rac1, p-Limk1, and p-Cofilin in the relative inhibitor group were significantly lower than those in the control group (p < 0.05), and the proliferation and migration levels in the control group and Rac1 overexpression group were significantly higher than those in the Rac1, Limk1, and Cofilin inhibitor group (p < 0.05). In conclusion, the decreased phosphorylation of the Rac1/Limk1/Cofilin signaling pathway in HSCR could inhibit the proliferation and migration of SH-SY5Y cells, and this might be associated with the pathogenesis of HSCR.

The Diagnostic and Prognostic Value of LIMK1/2: A Pan-Cancer Analysis.

BACKGROUND: LIM Kinase 1/2 (LIMK1/2) is a member of a conserved gene family of serine/threonine kinases. OBJECTIVE: This study aims to unravel the diagnostic and prognostic value of LIMK1/2 in pan-cancer. METHODS: A total of 33 cancer types with 11,057 samples from the TCGA database were downloaded. The immune subtypes C1 to C6 of cancers were indicated by previous studies. The ESTIMATE algorithm was used to calculate the infiltrating levels of immune cells and stromal cells. Cancer stemness was computed by DNAss and RNAss. Drug response was estimated using the CellMiner database. The functional enrichment analysis was performed by the Gene Set Enrichment Analysis method. RESULTS: LIMK1 is highly upregulated in multiple cancer tissues compared with normal tissues. LIMK2 is also significantly dysregulated in various cancer types. Abnormally expressed LIMK1 and LIMK2 can effectively predict the overall survival of different cancer patients. Further analyses were conducted to reveal the correlation between LIMK1/2 expression and immune subtype, immune scores, cancer stemness, tumor mutation burden, drug response, and functional enrichment analysis. CONCLUSION: The results provide a comprehensive insight into LIMK1 and LIMK2 in multiple cancers and as novel therapeutic targets for cancer treatment.

Mex-3 RNA binding MEX3A promotes the proliferation and migration of breast cancer cells via regulating RhoA/ROCK1/LIMK1 signaling pathway.

Breast cancer has been known as cancer with high mortality rates. It has been studied that MEX3A (Mex-3 RNA Binding Family Member A) is involved in carcinogenesis by accelerating cancer proliferation and migration. Therefore, this research aimed to study how MEX3A regulates the biological behaviors of breast cancer. Firstly, we used GEPIA and KM-plotter databases to evaluate MEX3A expression in human breast cancer tissue compared to adjacent normal tissue. Immunohistochemistry was employed to assess MEX3A protein expression in clinical specimens. MEX3A mRNA expression level was assessed through quantitative real-time PCR (RT-qPCR). Western blotting was used to detect protein expression. Moreover, Cell Count Kit-8 (CCK-8) assay, wound healing assay and transwell invasion assay were used to determine the proliferation, migration and invasion of breast cancer cells, respectively. Our study found that MEX3A expression level was much higher in human breast cancer tissues as compared to adjacent normal tissues. Similarly, breast cancer cell lines showed higher expression of MEX3A as compared to the normal breast cells. This higher expression of MEX3A was linked with the poor survival of breast cancer. Moreover, we found that overexpression of MEX3A stimulated proliferation and migration in the breast cancer cells. However, inhibition of MEX3A significantly reduced the proliferation and migration of breast cancer cells. In addition, we determined that MEX3A could activate RhoA/ROCK1/LIMK1 signaling in the breast cancer cells. Overall, our study concluded that MEX3A promotes its migration and proliferation in breast cancer cells via modulating RhoA/ROCK1/LIMK1 signaling pathway.

LIM-Kinases in Synaptic Plasticity, Memory, and Brain Diseases.

Learning and memory require structural and functional modifications of synaptic connections, and synaptic deficits are believed to underlie many brain disorders. The LIM-domain-containing protein kinases (LIMK1 and LIMK2) are key regulators of the actin cytoskeleton by affecting the actin-binding protein, cofilin. In addition, LIMK1 is implicated in the regulation of gene expression by interacting with the cAMP-response element-binding protein. Accumulating evidence indicates that LIMKs are critically involved in brain function and dysfunction. In this paper, we will review studies on the roles and underlying mechanisms of LIMKs in the regulation of long-term potentiation (LTP) and depression (LTD), the most extensively studied forms of long-lasting synaptic plasticity widely regarded as cellular mechanisms underlying learning and memory. We will also discuss the involvement of LIMKs in the regulation of the dendritic spine, the structural basis of synaptic plasticity, and memory formation. Finally, we will discuss recent progress on investigations of LIMKs in neurological and mental disorders, including Alzheimer's, Parkinson's, Williams-Beuren syndrome, schizophrenia, and autism spectrum disorders.

LIMK2 is required for membrane cytoskeleton reorganization of contracting airway smooth muscle.

Airway smooth muscle (ASM) has developed a mechanical adaption mechanism by which it transduces force and responds to environmental forces, which is essential for periodic breathing. Cytoskeletal reorganization has been implicated in this process, but the regulatory mechanism remains to be determined. We here observe that ASM abundantly expresses cytoskeleton regulators Limk1 and Limk2, and their expression levels are further upregulated in chronic obstructive pulmonary disease (COPD) animals. By establishing mouse lines with deletions of Limk1 or Limk2, we analyse the length-sensitive contraction, F/G-actin dynamics, and F-actin pool of mutant ASM cells. As LIMK1 phosphorylation does not respond to the contractile stimulation, LIMK1-deficient ASM develops normal maximal force, while LIMK2 or LIMK1/LIMK2 deficient ASMs show approximately 30% inhibition. LIMK2 deletion causes a significant decrease in cofilin phosphorylation along with a reduced F/G-actin ratio. As LIMK2 functions independently of cross-bridge movement, this observation indicates that LIMK2 is necessary for F-actin dynamics and hence force transduction. Moreover, LIMK2-deficient ASMs display abolishes stretching-induced suppression of 5-hydroxytryptamine (5-HT) but not acetylcholine-evoks force, which is due to the differential contraction mechanisms adopted by the agonists. We propose that LIMK2-mediated cofilin phosphorylation is required for membrane cytoskeleton reorganization that is necessary for ASM mechanical adaption including the 5-HT-evoked length-sensitive effect.

Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/- mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/- TNFα and +/- LIM kinase inhibitors (LIMKi). In silico modeling of TNFα-Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin-tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng-siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/-, and MLEC+/- compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.