Role In Cytoskeletal Rearrangement Research Articles

Cutting Edge: DOCK8 Regulates a Subset of Dendritic Cells That Is Critical for the Development of Experimental Autoimmune Encephalomyelitis.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor with an essential role in cytoskeletal rearrangement, cell migration, and survival of various immune cells. Interestingly, DOCK8-deficient mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE). To understand if EAE resistance in these mice results from an alteration in dendritic cell (DC) functions, we generated mice with conditional deletion of DOCK8 in DCs and observed attenuated EAE in these mice compared with control mice. Additionally, we demonstrated that DOCK8 is important for the existence of splenic conventional DC2 and lymph node migratory DCs and further established that migratory DC, rather than resident DC, are essential for the generation and proliferation of pathogenic T cell populations upon immunization with myelin Ag in adjuvant. Therefore, our data suggest that limiting migratory DCs through DOCK8 deletion and possibly other mechanisms could limit the development of CNS autoimmunity.

Down-regulation of ERMN expression in relapsing remitting multiple sclerosis.

Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and neurodegeneration in the central nervous system (CNS). Although the exact etiology of MS is still unclear, both genetic and environmental elements are regarded as causative factors. Environmental factors can induce a cascade of events in immune system leading to neuronal death and nerve demyelination. This paper aims to compare the peripheral transcript levels of Ermin (ERMN) (a gene with putative role in cytoskeletal rearrangements during myelinogenesis) and Listerin E3 Ubiquitin Protein Ligase 1 (LTN1) (a gene with functions in regulating innate immune system) between relapsing-remitting MS (RR-MS) patients and healthy controls. The results showed a significant decrease in ERMN expression (p = 0.022); whereas, no significant difference was detected in LTN1 expression between two groups (p = 0.935). The reduction in ERMN expression in leukocytes could be the cause of demyelinating process in RR-MS patients. Current findings might also have practical importance in prognosis and targeted therapies.

Parameters that define Suppressor of IKK epsilon (SIKE): cytoskeletal protein interactions revealed through determination of binding affinities and SIKE's dimer interface

Upon pathogen challenge multiple receptors both inside and on the surface of the cell, recognize pathogen associated molecular patterns (PAMPs) and initiate the production of proinflammatory, antiviral, and apoptotic responses. Pathways converge at key hubs that serve to amplify and regulate the signals, and are often responsible for determining the downstream response. TANK Binding Kinase 1 (TBK1) serves as a catalytic hub in the antiviral TLR3 mediated innate immune pathway. Suppressor of IKK epsilon (SIKE) is a recently identified high affinity alternative substrate of TBK1. It was initially found to inhibit TBK1 activation of type 1 interferon production. Upon subsequent study, it was found that SIKE was phosphorylated at six serine residues by TBK1. This phosphorylation of SIKE corresponds to the activation of the antiviral response, and releases SIKE from the SIKE:TBK1 interaction. The primary function of SIKE remains unknown. Examination of SIKE's interaction network has established direct interactions with cytoskeletal proteins including tubulin and α‐actinin. Migration assays have shown that chronic myelogenous leukemia (CML) cells in which SIKE has been knocked out migrate at a slower rate. Together, these studies suggest that SIKE plays a role in cytoskeletal rearrangements associated with innate immune responses such as migration and phagocytosis. The goal of this study is to define the interaction surface of SIKE as well as the binding affinities associated with these interactions. A quartz crystal microbalance with dissipation (QCM‐D) assay was developed to obtain binding affinities for the SIKE:cytoskeletal protein complexes. A gold sensor was utilized and functionalized with protein G and α‐His antibody to which 6xHis‐SIKE was immobilized. Increasing concentrations of binding partner were flowed over the chip to develop a binding curve. Prior to examining SIKE:cytoskeletal protein interactions, SIKE's oligomeric state was defined by crosslinking studies. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) shows that SIKE is a dimer. Chemical crosslinking followed by tandem mass spectrometry was employed to determine the dimeric interface of the SIKE dimer. BS3 was used in an excess of 20 and 100X. Data were analyzed with Mascot using xComb strategy. Residues 13, 66, 68, 110, and 169 were found to be accessible to BS3 modification, while in the dimer only residues 13, 110, 119, and 120 were found to be accessible. Residues 110 and 119 are found in multiple crosslinked peptides in both dimer and monomer samples. Residues 178 and 195 were found in crosslinked peptides unique to the dimer. These data have been applied to SIKE dimer models, generated by computational docking experiments to select for models consistent with the crosslinking restraints. These data define the dimer interface of SIKE as well as the surfaces available for SIKE cytoskeletal protein interactions. Together, these studies begin to reveal parameters that determine SIKE's interactions and the protein surfaces available to mediate these interactions.Support or Funding InformationStudies were funded by USD SURE, Beckman Foundation, NIH‐NIAID R21.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Role of the Non-Receptor Tyrosine Kinase Fes in Cancer

Non receptor protein tyrosine kinases are targets in the treatment of a number of diseases. This review focuses on the role of Fes tyrosine kinase and on the design of inhibitors of this protein. Fes and its homologously related protein Fer are the only two members of a distinct class of non receptor tyrosine kinases and they seem to play a role in cytoskeletal rearrangements and inside-out signaling associated with receptor-ligand, cell-matrix and cell-cell interactions. The knowledge of the three dimensional structure of this protein, in fact, has informed drug design, while at the same time it has helped to shed some light on the molecular mechanism at the basis of kinase activation and functions.

Eight full‐length abelson related gene (Arg) isoforms are constitutively expressed in caki‐1 cell line and cell distribution of two isoforms has been analyzed after transfection

The human Arg (Abl2) nonreceptor tyrosine kinase has a role in cytoskeletal rearrangements by its C-terminal F-actin- and microtubule-binding sequences. We have previously identified Arg transcripts with different 5'- and 3'-ends, named respectively long and short 1A and 1B (1AL, 1AS, 1BL, 1BS) and long and short C-termini (CTL and CTS), that have different expression patterns in various cell types. The combination of the different ends permits to predict eight putative full-length Arg transcripts and corresponding proteins. By Reverse Transcription-Long PCR we show here that all eight full-length transcripts are endogenously expressed in Caki-1 cells and the two bands, approximately 10 kDa different, shown by 1-D Western blots of Hek293T and Caki-1 lysates correspond to the full-length Arg protein isoforms with different C-termini. 2-D Western blot analysis evidenced different high molecular weight and slight acidic specific spots in Hek293T and Caki-1 lysates. The cellular localization of two Arg isoforms (1BLCTL and 1BLCTS) transfected in Caki-1 and Hek293T cells was cytoplasmic, and some differences in cytoskeleton interactions have been evidenced. Moreover, in Hek293T cells only the transfected 1BLCTS isoform gives rise to a large intracytoplasmic cylindrical structure containing phalloidin-positive amorphous actin aggregates. The presence of eight full-length Arg isoforms with different cellular expression may imply a diverse functional role in normal and neoplastic cells.

Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation

Sphingosine-1-phosphate (S1P) plays a vital role in cytoskeletal rearrangement, development, and apoptosis. Sphingosine kinase-1 (SphK1), the key enzyme catalyzing the formation of S1P, mediates ischaemic preconditioning. Ischaemic postconditioning (POST) has been shown to protect hearts against ischaemia/reperfusion injury (IR). To date, no studies have examined the role of SphK1 in POST. Wild-type (WT) and SphK1 null (KO) mouse hearts were subjected to IR (45 min of global ischaemia and 45 min of reperfusion) in a Langendorff apparatus. Left ventricular developed pressure (LVDP), maximum velocity of increase or decrease of LV pressure (+/-dP/dtmax), and LV end-diastolic pressure (LVEDP) were recorded. Infarction size was measured by 1% triphenyltetrazolium chloride staining. POST, consisting of 5 s of ischaemia and 5 s of reperfusion for three cycles after the index ischaemia, protected hearts against IR: recovery of LVDP and +/-dP/dtmax were elevated; LVEDP was decreased; infarction size (% of risk area) was reduced from 40 +/- 2% in the control group to 29 +/- 2% of the risk area in the POST group (P < 0.05, n = 4 per group). Phosphorylation of Akt and extracellular signal-regulated kinases detected by Western blotting was increased at 10 min of reperfusion. The protection induced by POST was abolished in KO hearts. Infarction size in KO hearts (57 +/- 5%) was not different from the KO control group (53 +/- 5% of risk area, n = 4, P = NS). A short period of ischaemic POST protected WT mouse hearts against IR. The cardiac protection induced by POST was abrogated in SphK1-KO mouse hearts. Thus, SphK1 is critical for successful ischaemic POST.

Phospholipase D1 Regulates Cell Migration in a Lipase Activity-independent Manner

Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.

Glycogen Synthase Kinase 3- and Extracellular Signal-Regulated Kinase-Dependent Phosphorylation of Paxillin Regulates Cytoskeletal Rearrangement

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.

Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actin-rich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis.

Current Topics in Pharmacological Research on Bone Metabolism: Inhibitory Effects of Bisphosphonates on the Differentiation and Activity of Osteoclasts

Despite the extensive use of bisphosphonates (BPs) in the treatment of metabolic bone diseases associated with increased osteoclastic bone resorption, the precise mechanism of their action on bone metabolism is still unclear. To clarify at which stages of osteoclast differentiation and activation that BPs influence, we examined the osteoclasts generated from mononuclear precursors and osteoclasts in the calvaria by laser scanning confocal microscopy. The studies showed that BPs inhibit lipopolysaccharide- or parathyroid hormone-induced osteoclast differentiation, fusion, attachment, actin ring formation, and activation and that both beta3 integrin and osteopontin have an important role in cytoskeletal rearrangements associated with cell attachment and resorption in osteoclasts.

Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon.

Hereditary spastic paraplegia (HSP) is characterized by the specific retrograde degeneration of the longest axons in the central nervous system, the corticospinal tracts. The gene most frequently involved in autosomal dominant cases of this disease, SPG4, encodes spastin, an ATPase belonging to the AAA family. AAA proteins are thought to exert their function by the energy-dependent rearrangement of protein complexes. The composite function of these proteins is directed by their binding to regulatory factors and adaptor proteins that target their activity into specific pathways in vivo. We previously found that overexpressed spastin interacts dynamically with microtubules and displays microtubule-severing activity. Here, we demonstrate that spastin is enriched in cell regions containing dynamic microtubules. During cell division spastin is found in the spindle pole, the central spindle and the midbody, whereas in immortalized motoneurons it is enriched in the distal axon and the branching points. Furthermore, spastin interacts with the centrosomal protein NA14, and co-fractionates with gamma-tubulin, a centrosomal marker. Deletion of the region required for binding to NA14 disrupts spastin interaction with microtubules, suggesting that NA14 may be an important adaptor to target spastin activity at the centrosome. These data strongly argue that spastin plays a role in cytoskeletal rearrangements and dynamics, and provide an attractive explanation for the degeneration of motor axons in HSP.

Calcium signalling during cell interactions with bacterial pathogens

The ability of a pathogenic microorganism to cause a disease is conditioned by its ability to colonise a given niche and implicates the expression of specific determinants, i.e. virulence factors, that allow the pathogen to adhere to or to invade epithelial cells. Diseases may be induced by bacteria that replicate extracellularly and alter the epithelial mucosa by producing toxins. Ca 2+ signalling has been implicated in various steps of bacterial infection. Bacterial toxins can induce an increase in free cytosolic Ca 2+ in host cells, itself required for the toxin-mediated effects. Such toxins, by diffusing in the extracellular media, can act at a distance from the site of infection and have a global effect on the integrity of the epithelium by promoting the expression of pro-inflammatory cytokines. Independent on toxins, bacteria can induce Ca 2+ responses that play a role in cytoskeletal rearrangements required for cell binding or internalisation of the microorganism. In some instances, invasion of the epithelium may be followed by bacterial access to deeper tissue, dissemination to other organs, and sometimes persistence in host cells in a parasitic-like mode. Such strategies underline the pathogen abilities to control innate defence cells such as professional phagocytes, and may implicate the diversion of Ca 2+-dependent cellular processes that normally result in killing of the ingested bacteria. Finally, bacterial pathogens can also induce the cell release of ATP, a Ca 2+ agonist, that may expand bacterial cell signalling by a paracrine or autocrine route, leading to enhanced colonisation or enhanced host cell responses to the invading microorganism.

Incorporation of MAP Kinases Into the Platelet Cytoskeleton

Erk1 (p44) and erk2 (p42) mitogen-activated protein (MAP) kinases are activated in agonist-stimulated platelets, although their role(s) in the activation process is unknown. In the present study, erk1, erk2 and the phosphorylated forms of both enzymes became associated with the contractile cytoskeleton in thrombin-stimulated platelets. Enzyme incorporation was accompanied by an increase in MAP kinase activity in the cytoskeleton, which was inhibited by PD98059. Pretreatment of the platelets with the arginine–glycine–aspartic acid–serine (RGDS) polypeptide enhanced both the cytoskeletal association and the enzyme activity, but cytochalasin D had no significant effect. Platelets from a patient with Glanzmann's thrombasthenia lack the α IIbβ 3 integrin and form only a rudimentary cytoskeleton, however, this cytoskeleton is enriched with both erk1 and erk2. These data suggest either that MAP kinases play a role in cytoskeletal rearrangement or that the cytoskeleton act as a frame to align MAP kinases with substrates in a highly integrated signal transduction pathway.

A Role for Wiskott-Aldrich Syndrome Protein in T-cell Receptor-mediated Transcriptional Activation Independent of Actin Polymerization

Wiskott-Aldrich syndrome protein (WASP) plays a key role in cytoskeletal rearrangement and transcriptional activation in T-cells. Recent evidence links WASP and related proteins to actin polymerization by the Arp2/3 complex. To study whether the role of WASP in actin polymerization is coupled to T-cell receptor (TCR)-mediated transcriptional activation, we made a series of WASP deletion mutants and tested them for actin co-localization, actin polymerization, and transcriptional activation of NFAT. A WASP mutant with a deletion in the C-terminal region (WASPDeltaC) that is defective in actin polymerization potentiated NFAT transcription following TCR activation by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol 12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not inhibit NFAT activation. Finally, by analyzing a series of WASP double-domain deletion mutants, we determined that the WASP homology-1 domain is responsible for NFAT transcriptional activation. Our results suggest that WASP activates transcription following TCR stimulation in a manner that is independent of its role in Arp2/3-directed actin polymerization.

Effects of melatonin on microtubule assembly depend on hormone concentration: Role of melatonin as a calmodulin antagonist

Melatonin may play a key role in cytoskeletal rearrangements through its calmodulin antagonism. In the present work, we tested this hypothesis by studying melatonin effects on both microtubule polymerization in vitro and cytoskeletons in situ. Microtubule assembly is a dynamic process inhibited by Ca2+/calmodulin. Calmodulin antagonists prevent the inhibition by binding to Ca(2+)-activated calmodulin, thus causing microtubule enlargement. In the presence of calmodulin (5 microM) and CaCl2 (1 mM), polymerization at equilibrium was inhibited by 40%. Complete reversal of the Ca2+/calmodulin effect on microtubules was observed with 10(-9) M melatonin or with 10(-5) M trifluoperazine or 1 microgram/ml of compound 48/80. In the absence of Ca2+/calmodulin, melatonin at 10(-5) M inhibited tubulin polymerization like 10(-4) M trifluoperazine does. Melatonin effects on microtubule assembly at both nanomolar and micromolar ranges were corroborated in cytoskeletons in situ. Therefore, it is suggested that at a low concentration (10(-9) M), cytoskeletal melatonin effects are mediated by its antagonism to Ca2+/calmodulin. At a higher concentration (10(-5) M), non-specific binding of melatonin to tubulin occurs, thus overcoming the melatonin antagonism to Ca2+/calmodulin. The results support the hypothesis that under physiological conditions, melatonin synchronizes different body rhythms through cytoskeletal rearrangements mediated by its calmodulin antagonism.