LIMK, Cofilin 1 and actin dynamics involvement in fear memory processing.

Long-term memory has been associated with morphological changes in the brain, which in turn tightly correlate with changes in synaptic efficacy. Such plasticity is proposed to rely on dendritic spines as a neuronal canvas on which these changes can occur. Given the key role of actin cytoskeleton dynamics in spine morphology, major regulating factors of this process such as Cofilin 1 (Cfl1) and LIM kinase (LIMK), an inhibitor of Cfl1 activity, are prime molecular targets that may regulate dendritic plasticity. Using a contextual fear conditioning paradigm in mice, we found that pharmacological induction of depolymerization of actin filaments through the inhibition of LIMK causes an impairment in memory reconsolidation, as well as in memory consolidation. On top of that, Cfl1 activity is inhibited and its mRNA is downregulated in CA1 neuropil after re-exposure to the training context. Moreover, by pharmacological disruption of actin cytoskeleton dynamics, the process of memory extinction can either be facilitated or impaired. Our results lead to a better understanding of the role of LIMK, Cfl1 and actin cytoskeleton dynamics in the morphological and functional changes underlying the synaptic plasticity of the memory trace.

FHOD3 promotes carcinogenesis by regulating RhoA/ROCK1/LIMK1 signaling pathway in medulloblastoma.

PURPOSE: Medulloblastoma (MB) is a malignant brain disease in young children. The overall survival of MB patients is disappointing due to absence of effective therapeutics and this could be attributed to the lack of molecular mechanism underlying MB. FHOD3 was an important gene during cardio-genesis and was reported to promote cell migration in cancer. However, its role in MB is not clear to date. METHODS: RT-qPCR and IHC analysis were used to determine expression of FHOD3. Survival curve was drawn by K-M analysis. FHOD3 was knocked down by RNAi technology. The effects of FHOD3 on medulloblastoma cells were determined by CCK-8 assay, colony formation assay, transwell assay and FACs analysis. RESULTS: FHOD3 expression increased by 1.5 fold in tumor tissues compared to the control and IHC analysis further confirmed strong expression of FHOD3 in medulloblastoma tissues. Then higher FHOD3 expression was associated with shorter survival time in MB patients (13.0 months versus 43.8 months). In medulloblastoma cells such as Daoy and D283med, FHOD3 also displayed abundant expression. When FHOD3 was knocked down, the ability of cell proliferation and colony formation was reduced over greatly. The capability of cell migration and invasion was also inhibited significantly. However, cell apoptotic rate increased significantly reversely. Mechanistically, the phosphorylation level of RhoA, ROCK1, and LIMK1 was decreased when FHOD3 was knocked down but increased reversely when FHOD3 was over-expressed in Daoy cells. CONCLUSIONS: FHOD3 was associated with overall survival time in medulloblastoma patients and was essential to cell proliferation, growth and survival in medulloblastoma and might regulates activation of RhoA/ROCK1/LIMK1 signaling pathway.

Agonist Effects of Propranolol on Non-Tumor Human Breast Cells.

The ß-blocker propranolol (PROP) has been proposed as a repurposed treatment for breast cancer. The similarity of action between ß-agonists and antagonists found on breast cells encouraged us to compare PROP and isoproterenol (ISO, agonist) signaling pathways on a human breast cell line. Cell proliferation was measured by cell counting and DNA-synthesis. Cell adhesion was measured counting the cells that remained adhered to the plastic after different treatments. Changes in actin cytoskeleton were observed by fluorescence staining and Western Blot. ISO and PROP caused a diminution of cell proliferation and an increase of cell adhesion, reverted by the pure ß-antagonist ICI-118551. ISO and PROP induced a reorganization of actin cytoskeleton increasing F-actin, p-COFILIN and p-LIMK. While ISO elicited a marked enhancement of cAMP concentrations and an increase of vasodilator-stimulated phosphoprotein (VASP) and cAMP response element-binding protein (CREB) phosphorylation, PROP did not. Clathrin-mediated endocytosis inhibition or ß-arrestin1 dominant-negative mutant abrogated PROP-induced cell adhesion and COFILIN phosphorylation. The fact that PROP has been proposed as an adjuvant drug for breast cancer makes it necessary to determine the specific action of PROP in breast models. These results provide an explanation for the discrepancies observed between experimental results and clinical evidence.

Cofilin-1 signaling mediates epithelial-mesenchymal transition by promoting actin cytoskeleton reorganization and cell-cell adhesion regulation in colorectal cancer cells.

Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-ß (siCofilin-1/TGF-ß) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-ß cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.

Role of Actin Cytoskeleton During Mammalian Sperm Acrosomal Exocytosis.

Mammalian sperm require to undergo an exocytotic process called acrosomal exocytosis in order to be able to fuse with the oocyte. This ability is acquired during the course of sperm capacitation. This review is focused on one aspect related to this acquisition: the role of the actin cytoskeleton. Evidence from different laboratories indicates that actin polymerization occurs during capacitation, and the detection of several actin-related proteins suggests that the cytoskeleton is involved in important sperm functions. In other mammalian cells, the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis but, at the same time, is necessary to prepare the cell to undergo regulated exocytosis. Thus, F-actin stabilizes structures generated by exocytosis and supports the physiological progression of this process. Is this also the case in mammalian sperm? This review summarizes what is currently known about actin and its related proteins in the male gamete, with particular emphasis on their role in acrosomal exocytosis.

Overexpressed Down Syndrome Cell Adhesion Molecule (DSCAM) Deregulates P21-Activated Kinase (PAK) Activity in an In Vitro Neuronal Model of Down Syndrome: Consequences on Cell Process Formation and Extension.

In humans, Down syndrome (DS) is caused by the presence of an extra copy of autosome 21. The most striking finding in DS patients is intellectual disability and the onset of Alzheimer's disease (AD)-like neuropathology in adulthood. Gene overdose is most likely to underlie both developmental impairments, as well as altered neuronal function in DS. Lately, the disruption of cellular signaling and regulatory pathways has been implicated in DS pathophysiology, and many of such pathways may represent common targets for diverse DS-related genes, which could in turn represent attractive therapeutical targets. In this regard, one DS-related gene Down Syndrome Cell Adhesion Molecule (DSCAM), has important functions in neuronal proliferation, maturation, and synaptogenesis. p21-associated kinases (PAKs) appear as a most interesting possibility for study, as DSCAM is known to regulate the PAKs pathway. Hence, in DS, overexpressed DSCAM could deregulate PAKs activity and affect signaling pathways that regulate synaptic plasticity such as dendritic spine dynamics and axon guidance and growth. In the present work, we used an immortalized cell line derived from the cerebral cortex of an animal model of DS such as the trisomy 16 (Ts16) fetal mouse (named CTb), and a similar cell line established from a normal littermate (named CNh), to study the effect of DSCAM in the PAKs pathway. The present study shows that DSCAM is overexpressed in CTb cells by approximately twofold, compared to CNh cells. Congruently, PAK1, as well as its downstream effectors LIMK and cofilin, stay phosphorylated for longer periods after DSCAM activation in the CTb cells, leading to an altered actin dynamics, expressed as an increased basal F/G ratio and reduced neurite growth, in the trisomic condition. The present work presents the correlation between DSCAM gene overexpression and a dysregulation of the PAK pathway, resulting in altered morphological parameters of neuronal plasticity in the trisomic cell line, namely decreased number and length of processes.

Distribution of LIM domain kinase 1 in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum of the App/PS+/- mice.

LIM domain kinase 1 (LIMK1), an actin-binding kinase, can phosphorylate and inactivate its substrates, and can regulate long-term memory and synaptic plasticity. Both ß-amyloid precursor protein (App) and presenilin (PS) are functional degeneration factors during early neuronal development, and are considered as potential factors that contribute to the development of Alzheimer's disease (AD). However, hardly any information is available about the distribution and expression of LIMK1. Thus, using the App and PS deficient mice, the role of LIMK1 was demonstrated in the absence of App and PS. Our results showed that LIMK1 was present in the nerve fiber layer and external plexiform layer of the olfactory bulb, as well as in the mitral cells and Purkinje cells of the cerebellum in App and PS deficient mice. Additionally, LIMK1 was concentrated in the granule cell layer of the olfactory bulb and cerebellum and LIMK1 positive cells were located in the CA1 region of the hippocampus. Our study indicates that there is a connection between LIMK1 and AD in the mouse model of AD. This might explain neurological problems such as cerebellar ataxia, impaired long-term memory, and impaired synaptic plasticity observed in AD.

PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.

Bone morphogenetic protein 2 inhibits neurite outgrowth of motor neuron-like NSC-34 cells and up-regulates its type II receptor.

Bone morphogenetic proteins (BMPs) regulate several aspects of neuronal behavior. For instance, BMP-2 has the ability to modulate, either positively or negatively, the outgrowth of neuronal processes in diverse cell types. In Drosophila motor neurons, the BMP type II receptor (BMPRII) homolog wishful thinking plays crucial roles on neuromuscular synaptogenesis signaling through Smad-dependent and Smad-independent pathways. However, a role for BMP signaling at the vertebrate neuromuscular junction has not been addressed. Herein, we have analyzed the expression of BMPRII and the effect of BMP-2 during the morphological differentiation of motor neuron-like NSC-34 cells. Our data indicate that BMPRII is up-regulated and becomes accumulated in somas and growth cones upon motor neuronal differentiation. BMP-2 inhibits the differentiation of NSC-34 cells, an effect that correlates with activation of a Smad-dependent pathway, induction of the inhibitory Id1 transcription factor, and down-regulation of the neurogenic factor Mash1. BMP-2 also activates effectors of Smad-independent pathways. Remarkably, BMP-2 treatment significantly increases the expression of BMPRII. Our findings provide the first evidence to suggest a role for BMP pathways on the differentiation of motor neurons leading to successful assembly and/or regeneration of the vertebrate neuromuscular synapse.

Leukotrienes target F-actin/cofilin-1 to enhance alveolar macrophage anti-fungal activity.

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

MAP1B regulates axonal development by modulating Rho-GTPase Rac1 activity.

Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.