Promotes Actin Polymerization Research Articles

P2‐202: INHIBITION OF ACTIVITY‐DEPENDENT PROTEIN SYNTHESIS BY IL‐1β IN HIPPOCAMPAL DENDRITES

In rodent hippocampus, the inflammatory cytokine interleukin-1β (IL-1β) impairs memory and long-term potentiation (LTP), a major form of plasticity that depends on protein synthesis. We tested whether IL-1β inhibits protein synthesis in hippocampal neuron cultures following chemically-induced LTP (cLTP). Fluorescent-tagging using click-chemistry showed that IL-1β reduces the level of newly synthesized proteins in proximal dendrites of cLTP stimulated neurons. Relative to controls, in cLTP stimulated neurons IL-1β inhibited Akt/mTOR signaling, as well as the upregulation of GluA1, an AMPA receptor subunit, and LIMK1, a kinase that promotes actin polymerization. Notably, a novel TIR domain peptidomimetic (EM163) blocked both the activation of p38 and the suppression of cLTP-dependent protein synthesis by IL-1β. Our data support a model where IL-1β suppresses LTP directly in neurons by inhibiting mTOR-dependent translation

Abstract 209: Metastatic colonization in the perivascular niche: A mechanotransduction perspective

Abstract Metastases are responsible for majority cancer related deaths. Disseminated tumor cells (DTCs) gain a diverse array of metastatic traits in order to adapt to and survive within different microenvironments, making them very difficult to therapeutically target. Remarkably, regardless of the secondary organ, DTCs survive and thrive in perivascular niches. Using xenograft and syngeneic models of metastasis in mouse, we found that upon extravasation, neuronal cell adhesion molecule L1 (L1CAM) expressed by DTCs engages the vascular basal lamina in order to amplify integrin β1 and integrin linked kinase (ILK) signaling in DTCs. ILK promotes actin polymerization for cell spreading and subsequent activation of mechanotransduction effector transcription factors YAP and MRTF. We found that activation of YAP and MRTF coincides with DTCs ability to dislodge resident pericytes from the perivascular niche and is an essential step in the metastatic colonization regardless of dormancy state. Therefore, these results provided us a unifying molecular mechanism that governs cancer progression from metastatic seeding to overt colonization. Ongoing studies will determine how mechanotransduction and pericyte-DTC interactions can be manipulated to prevent metastatic colonization by potentiating the immune response and exposing their biophysical vulnerabilities. Citation Format: Ekrem Emrah Er, Maria Tello-Lafoz, Jing Hu, Morgan Huse, Joan Massagué. Metastatic colonization in the perivascular niche: A mechanotransduction perspective [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 209.

The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host⁻Pathogen Interactions and Innate Immunity.

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions.

LIM Kinase Inhibition Diminishes Hypertension and Vasoconstriction‐Induced Inward Remodeling in Mouse and Human Resistance Arteries

Vascular remodeling is an early biomarker of cardiovascular disease (CVD) related to structural changes in resistance arteries (RA). Arteriolar inward remodeling, characterized by a reduced luminal diameter that is associated with changes in the homeostasis of actin polymerization/depolymerization pathways, is posited to precede the development of hypertension. We hypothesize that inward remodeling involves changes in the actin cytoskeleton that facilitate cell motility and the rearrangement of vascular smooth muscle cells (VSMCs) around the vascular lumen. Moreover, we predict that inhibition of actin polymerization with a Lim kinase (LIMK) inhibitor would block the inward remodeling process associated with prolonged vasoconstriction and hypertension. We have previously reported in ex vivo experiments in rats that long‐term exposure of RA to vasoconstrictors induces vascular inward remodeling via activation of the Rho pathway, which in part promotes actin polymerization. We now demonstrate that VSMCs increase the inward remodeling ratio of filamentous to globular actin (F/G‐actin) in response to GPCR‐activation by a norepinephrine (NE) and angiotensin‐II (AngII). GPCR‐mediated Rho activates (LIMK), which subsequently phosphorylates and inactivates the actin severing enzyme, cofilin, thus effecting an increase in F/G‐actin ratios. In isolated rat cremasteric RA we have demonstrated that LIMK inhibition blocks the inward remodeling induced by of GPCR agonists and that this is coincident with a reduction in cofilin phosphorylation. To study the arterial remodeling process in vivo, we imaged the outer ear of a transgenic mouse line that expresses smooth muscle actin‐GFP in VSMCs by multiphoton microscopy. We treated these mice with AngII (1000ng/kg/min) for 14 days to induce vasoconstriction and hypertension. We imaged the same resistance artery within each ear before (Day 0) and after (Day 14) AngII infusion to study the progression of remodeling over time. Starting at day 0, one ear was injected every four days with a LIMK inhibitor (LIMKi), while the other ear received vehicle (control). We observed a significant inward remodeling in vessels of the control ear compared to those of the ear treated with LIMKi (p<0.05). We then assessed the effect of LIMKi in the inward remodeling process associated with ex vivo prolonged vasoconstriction in isolated human RA from omental tissues and studied by pressure myography under passive conditions before and after a 4h incubation in the presence of NE + AngII (control) or NE + AngII + LIMKi. Exposure to NE + AngII induced vascular inward remodeling that was completely ablated by presence of LIMKi and resulted in outward remodeling (p<0.05). These data indicate that LIMK plays a necessary role in the vasoconstriction‐hypertension vascular remodeling process that makes it a potential target for the treatment of CVDs associated with resistance artery inward remodeling.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic Actin Reorganization and Vav/Cdc42-Dependent Actin Polymerization Promote Macrophage Aggregated LDL (Low-Density Lipoprotein) Uptake and Catabolism.

Objective- During atherosclerosis, LDLs (low-density lipoproteins) accumulate in the arteries, where they become modified, aggregated, and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which leads to the progression of atherosclerosis. Therapeutic blockade of this process may delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment-the lysosomal synapse (LS)-aided by local actin polymerization to digest agLDL. Here, we investigated the specific regulators involved in actin polymerization during the formation of the LS. Approach and Results- We demonstrate in vivo that atherosclerotic plaque macrophages contacting agLDL deposits polymerize actin and form a compartment strikingly similar to those made in vitro. Live cell imaging revealed that macrophage cortical F-actin depolymerization is required for actin polymerization to support the formation of the LS. This depolymerization is cofilin-1 dependent. Using siRNA-mediated silencing, pharmacological inhibition, genetic knockout, and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and GEF (guanine nucleotide exchange factor) Vav in promoting actin polymerization during the formation of the LS and exclude a role for Rac1. Conclusions- These results highlight critical roles for dynamic macrophage F-actin rearrangement and polymerization via cofilin-1, Vav, and Cdc42 in LS formation, catabolism of agLDL, and foam cell formation. These proteins might represent therapeutic targets to treat atherosclerotic disease.

Transgelin 2 is required for embryo implantation by promoting actin polymerization.

Infertility has been a great challenge in reproductive medicine. At least 40% of human pregnancy losses are clinically unrecognized and occur because of embryo implantation failure. Identification of the proteins and biochemical factors involved in embryo implantation and that are essential for crosstalk between the embryo and uterus can further increase female fertility rates. The actin cytoskeleton and actin-binding proteins (ABPs) are of great importance for cell morphology and rearrangement, which is crucial for trophoblast adhesion and invasion. However, the research on ABPs in embryo implantation is insufficient. In this report, we found that transgelin (TAGLN)2 is highly expressed in mouse blastocyst trophoblasts. Notably, inhibition of mouse blastocyst trophoblast TAGLN2 by lentivirus-mediated RNA interference significantly impaired embryo adhesion and implantation ability. Further in vitro experiments demonstrated that TAGLN2 knockdown with small interfering RNA observably decreased the invasion and migration abilities of human trophoblast cells. Immunofluorescence colocalization and microscale thermophoresis analysis showed that TAGLN2 directly binds to actin. In addition, knockdown of TAGLN2 in trophoblast cells resulted in a remarkable reduction in F-actin rather than G-actin. Our findings reveal an unidentified role of TAGLN2 in regulation of trophoblast invasion and adhesion by promoting actin polymerization.-Liang, X., Jin, Y., Wang, H., Meng, X., Tan, Z., Huang, T., Fan, S. Transgelin 2 is required for embryo implantation by promoting actin polymerization.

Inhibition of LTP-Induced Translation by IL-1β Reduces the Level of Newly Synthesized Proteins in Hippocampal Dendrites.

In rodent hippocampus, the inflammatory cytokine interleukin-1β (IL-1β) impairs memory and long-term potentiation (LTP), a major form of plasticity that depends on protein synthesis. A better understanding of the mechanisms by which IL-1β impairs LTP may help identify targets for preventing cognitive deterioration. We tested whether IL-1β inhibits protein synthesis in hippocampal neuron cultures following chemically induced LTP (cLTP). Fluorescent-tagging using click-chemistry showed that IL-1β reduces the level of newly synthesized proteins in proximal dendrites of cLTP stimulated neurons. Relative to controls, in cLTP stimulated neurons, IL-1β inhibited Akt/mTOR signaling, as well as the upregulation of GluA1, an AMPA receptor subunit, and LIMK1, a kinase that promotes actin polymerization. Notably, a novel TIR domain peptidomimetic (EM163) blocked both the activation of p38 and the suppression of cLTP-dependent protein synthesis by IL-1β. Our data support a model where IL-1β suppresses LTP directly in neurons by inhibiting mTOR-dependent translation.

Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

The Rickettsia ∼1800-amino-acid autotransporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments of Sca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.

The cotton XLIM protein (GhXLIM6) is required for fiber development via maintaining dynamic F-actin cytoskeleton and modulating cellulose biosynthesis.

LIM domain proteins are cysteine-rich proteins, and are often considered as actin bundlers and transcription factors in plants. However, the roles of XLIM proteins in plants (especially in cotton) remain unexplored in detail so far. In this study, we identified a cotton XLIM protein (GhXLIM6) that is preferentially expressed in cotton fiber during whole elongation stage and early secondary cell wall (SCW) synthesis stage. The GhXLIM6-silenced transgenic cotton produces shorter fibers with thinner cell walls, compared with wild-type (WT). GhXLIM6 protein could directly bind F-actin and promote actin polymerization both invitro and invivo. It also acts as a transcription factor to suppress GhKNL1 expression through binding the PAL-box element of GhKNL1 promoter, and subsequently regulate the expression of CesA genes related to cellulose biosynthesis and deposition in SCWs of cotton fibers. The cellulose content in fibers of GhXLIM6RNAi cotton is lower than that in WT. Taken together, these data reveal the dual roles of GhXLIM6 in fiber development. On one hand, GhXLIM6 functions in fiber elongation through binding to F-actin to maintain the dynamic F-actin cytoskeleton. On the other hand, GhXLIM6 fine-tunes fiber SCW formation, probably through directly suppressing transcription of GhKNL1 to promote cellulose biosynthesis.

New slbo-Gal4 driver lines for the analysis of border cell migration during Drosophila oogenesis.

Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic6458 mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic6458 on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic6458 line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic6458, but not the new slbo-Gal4 drivers were used. These findings indicatetwf and chic interact and function antagonistically during BC migrationin Drosophila oogenesis.

An Intellectual Disability-Related Missense Mutation in Rac1 Prevents LTP Induction.

The small GTPase Rac1 promotes actin polymerization and plays a critical and increasingly appreciated role in the development and plasticity of glutamatergic synapses. Growing evidence suggests that disruption of the Rac1 signaling pathway at glutamatergic synapses contributes to Autism Spectrum Disorder/intellectual disability (ASD/ID)-related behaviors seen in animal models of ASD/ID. Rac1 has also been proposed as a strong candidate of convergence for many factors implicated in the development of ASD/ID. However, the effects of ASD/ID-related mutations in Rac1 itself have not been explored in neurons. Here, we investigate a recently reported de novo missense mutation in Rac1 found in an individual with severe ID. Our modeling predicts that this mutation will strongly inhibit Rac1 activation by occluding Rac1’s GTP binding pocket. Indeed, we find that this de novo mutation prevents Rac1 function and results in a selective reduction in synaptic AMPA receptor function. Furthermore, this mutation prevents the induction of long-term potentiation (LTP), the cellular mechanism underlying learning and memory formation. Together, our findings strongly suggest that this mutation contributes to the development of ID in this individual. This research demonstrates the importance of Rac1 in synaptic function and plasticity and contributes to a growing body of evidence pointing to dysregulation of actin polymerization at glutamatergic synapses as a contributing factor to ASD/ID.

Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle.

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase-inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.

A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits

SummaryMultiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

Endophilin A1 Promotes Actin Polymerization in Dendritic Spines Required for Synaptic Potentiation.

Endophilin A1 is a member of the N-BAR domain-containing endophilin A protein family that is involved in membrane dynamics and trafficking. At the presynaptic terminal, endophilin As participate in synaptic vesicle recycling and autophagosome formation. By gene knockout studies, here we report that postsynaptic endophilin A1 functions in synaptic plasticity. Ablation of endophilin A1 in the hippocampal CA1 region of mature mouse brain impairs long-term spatial and contextual fear memory. Its loss in CA1 neurons postsynaptic of the Schaffer collateral pathway causes impairment in their AMPA-type glutamate receptor-mediated synaptic transmission and long-term potentiation. In KO neurons, defects in the structural and functional plasticity of dendritic spines can be rescued by overexpression of endophilin A1 but not A2 or A3. Further, endophilin A1 promotes actin polymerization in dendritic spines during synaptic potentiation. These findings reveal a physiological role of endophilin A1 distinct from that of other endophilin As at the postsynaptic site.

Dynamic Actin Reorganization and Vav/Cdc42‐dependent Actin Polymerization Promote Macrophage Aggregated LDL Uptake and Catabolism

During atherosclerosis, low density lipoproteins (LDL) accumulate in the arteries, where they become modified, aggregated and retained. Such deposits of aggregated LDL (agLDL) can be recognized by macrophages, which attempt to digest and clear them. AgLDL catabolism promotes internalization of cholesterol and foam cell formation, which is an important component of atherosclerosis. Characterization and therapeutic blockade of this process will likely delay disease progression. When macrophages interact with agLDL in vitro, they form a novel extracellular, hydrolytic compartment, the lysosomal synapse, aided by local actin polymerization to digest agLDL. Here, we demonstrate in vivo that atherosclerotic plaque macrophages contacting cholesterol rich LDL deposits polymerize actin and form a strikingly similar compartment. Live cell imaging revealed macrophage cortical F‐actin depolymerization driven by PIP2 hydrolysis occurs in parallel to actin polymerization at the lysosomal synapse and is required for its formation. Using siRNA‐mediated silencing, pharmacological inhibition, genetic knockout and stable overexpression, we elucidate key roles for Cdc42 Rho GTPase and guanine nucleotide exchange factor Vav in promoting actin polymerization at the lysosomal synapse, and we rule out roles for Rac1 and Rac2. These data indicate that dynamic actin rearrangement and polymerization via Vav and Cdc42 aids compartment formation, catabolism of agLDL and promotes foam cell formation.Support or Funding InformationThe project described was supported by National Institutes of Health grants R37‐DK27083 and R01‐HL093324. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. R.K.S. is an American Heart Association Stanley Stahl Postdoctoral Fellow (ID: 15POST22990022).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Ubiquitination of Profilin‐1 in Hypoxia‐Induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a chronic illness characterized by increased pressure in the pulmonary arteries due to the thickening and constriction of the arterial walls. Vascular thickening is in part due to proliferation of smooth muscle cells. The ubiquitin proteasome system is an important regulator of cellular proliferation that alters protein stability and expression by modifying lysines (K) within target proteins with ubiquitin moieties. Addition of ubiquitin can alter activity of a protein or mark them for degradation. We hypothesized that hypoxia‐induced changes in ubiquitination impact PH development. Mass spectrometry (MS) was used to screen changes in ubiquitination of proteins in lung tissue from mice exposed to chronic hypoxia (10% O2) for 3 weeks. Profilin‐1, a mediator of actin‐polymerization, had decreased ubiquitination on K54 (Fold change −1.86) and K126 (Fold change −1.40). Profilin‐1 is known to play a role in cellular proliferation and its overexpression leads to increased wall thickness in aortas of spontaneously hypertensive rats. However, its role in PH is not well characterized. Proflin‐1 promotes actin polymerization by binding directly to ADP‐actin and catalyzing nucleotide exchange. Actin polymerization plays a key role in cellular proliferation, cell motility, and muscle contraction. We observed no difference in profilin‐1 protein levels in hypoxia‐exposed human pulmonary artery smooth muscle cells (HPASMC) and lungs from chronically hypoxia‐exposed mice when compared to normoxic controls (P>0.05). This lack of an effect of ubiquitination on protein levels suggests that Profilin‐1 ubiquitination could regulate its activity rather than stability. Preliminary fractionate on results show increased association of Profilin‐1 and G‐actin in hypoxia. Furthermore, disrupting actin polymerization using actin polymerization inhibitors decreased HPASMC proliferation. Therefore, our results indicate that reduced levels of ubiquitinated Profilin‐1 may promote actin polymerization which increases proliferation and contributes to PH. In addition, they suggest that changes in protein ubiquitination may contribute to PH progression.Support or Funding InformationThis work was supported in part by Merit Review funding from the Department of Veterans Affairs, Office of Research and Development (1I01BX001910 to CMH), by NIH Grant R01HL102167 (to CMH and RLS), and by support from NIH T32 HL076118 and American Heart Association 16POST30930007 (to BEW). MS work was performed by the Emory Integrated Proteomics Core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of inward arterial remodeling is ameliorated in vivo by inhibition of actin polymerization dynamics in a mouse model of hypertension

Hypertension is associated with structural changes to the resistance vasculature leading to a reduction in resistance artery diameter. This process is termed inward remodeling and is defined as a reduction in lumenal diameter under passive conditions. We posit that inward remodeling involves dynamic changes to the actin cytoskeleton that facilitate the movement and rearrangement of vascular smooth muscle cells around a smaller vascular lumen. Ex vivo, prolonged exposure to vasoconstrictors has been demonstrated to induce remodeling via g‐protein coupled receptors (GPCR) that activate the Rho signaling pathway and subsequently promote actin polymerization. We report here that vascular smooth muscle cells, propagated on nanopatterned culture‐ware to maintain their contractile phenotype, increase their ratio of filamentous (F) to globular (G)‐actin in response to GPCR activation by Norepinephrine and Angiotensin (Ang) II. One of the enzymes in the Rho signaling pathway implicated in the increase in F/G‐actin following GPCR activation is Lim Kinase, which phosphorylates and inactivates the actin severing enzyme, cofilin. In isolated rat cremasteric arteries we demonstrate that inhibition of Lim Kinase blocks inward remodeling in the presence of GPCR agonists and this is coincident with a reduction in cofilin phosphorylation. To examine the process in vivo, we utilized a transgenic mouse line that expresses smooth muscle actin (SMA)‐GFP in vascular smooth muscle cells to measure proximal arterial diameter noninvasively within the outer ears using confocal microscopy. Exposure of these mice to 1000 ng/kg/min of Ang II for two weeks resulted in a significant increase in mean arterial blood pressure. Both ears were imaged prior to Ang II infusion (Day 0) and after 14 days of infusion to assess inward remodeling. Starting at day 0, the right ear was injected with a bolus of a Lim Kinase inhibitor (10 uM) and the left ear was injected with vehicle. Injections were repeated every 4 days for a total of four injections. The vehicle exposed ears significantly inwardly remodeled in contrast to those exposed to the Lim Kinase inhibitor (Vehicle diameter = −15.95 % +/− 1.79 versus Lim Kinase inhibitor diameter = −1.75% +/− 1.78, P<0.05). These data demonstrate that in vivo, Lim Kinase plays a necessary role in inward remodeling, and thus merits additional study as a potential therapeutic target in cardiovascular pathologies associated with inward remodeling.Support or Funding InformationNIH‐HL‐088105 to LAMThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

23.1 UNDERSTANDING THE ROLE OF SCHIZOPHRENIA/AUTISM GENES IN CORTICAL DEVELOPMENT

BackgroundThe fidelity of neocortical development is dependent on the highly polarized morphology of the neuroepithelial stem cell (NSC) within the embryonic brain. NSCs project long processes to the pial surface along which newborn neurons migrate to establish the cortical plate. Perturbation of NSC morphology prevents neuronal migration into the emerging cortical layers, leading to cortical malformations. Disruption of the laminar architecture due to failed neuronal migration is thought to contribute to the etiology of schizophrenia and autism. Therefore, elucidating the signaling events that precisely control NSC morphology is essential to our understanding of corticogenesis and the aberrant processes that contribute to neuropsychiatric disorders.Maintenance of NSC morphology and function requires the formation of cadherin-based cell-cell adhesion (adherens junctions) between NSCs and loss of junctional integrity results in failed neuronal migration. Junctional stability is critically dependent on the closely apposed actin cytoskeleton and the actin remodeling protein Cyfip1 known to promote actin polymerization. Cyfip1 has been implicated in schizophrenia and autism and its loss results in cortical malformations. However, the molecular mechanisms governing Cyfip1 activity in NSCs are poorly understood.MethodsIn this study we investigate the signaling mechanisms that regulate Cyfip1 activity in the developing mouse cortex using both gain- and loss-of-function approaches. Short interfering RNAs or cDNA expression constructs were electroporated, in utero, into the embryonic day 12 mouse cortex. Phenotypic analysis was then performed several days later.ResultsHere we identify the netrin/RGM receptor, Neogenin, as a direct binding partner for Cyfip1. We provide evidence that Neogenin is a critical upstream regulator of Cyfip1 activity during corticogenesis and is therefore a key component of NSC junctions. We show that blocking Neogenin/Cyfip1 interactions in the embryonic mouse cortex results in NSC junctional collapse and severe perturbation of the emerging cortical architecture due to aberrant neuronal migration. Our study therefore reveals that Neogenin’s interaction with Cyfip1 is essential for NSC morphology and function.DiscussionIn conclusion, we have identified a novel signaling pathway that governs the development of the neocortex. The emergence of neuronal migration defects and cortical malformations when Neogenin-Cyfip1 interactions are prevented emphasizes the fundamental role of this interaction in establishing the correct cortical architecture. Intriguingly, mutations in the Neogenin gene have recently been linked to autism. Therefore, our study implicates the Neogenin/Cyfip pathway in the etiology of neuropsychiatric disorders.

MDIA1 Senses Both Force and Torque during F-actin Filament Polymerisation

Formins play a crucial role in regulating actin polymerization. A formin dimer binds to the barbed end of F-actin through its ring-like dimerized FH2 domains and recruits G-actin monomers through its proline-rich FH1 domains in a profilin dependent manner. Formins are often subject to mechanical stretch due to actomyosin contraction; therefore its function has been believed to be dependent on mechanical force. We have recently investigated the effects of force on formin-dependent actin polymerization. In both the presence and the absence of profilin, we observed that force could strongly promote actin polymerization, which strictly required that the actin filament could freely rotate around the formin. These results revealed that formins senses not only tensile force but also torque in the filament. Together, these results provide important new insights into the mechanosensing functions of formin-dependent actin organization in living cells.

Cortactin recruits FMNL2 to promote actin polymerization and endosome motility in invadopodia formation

Recently, invadopodia have been increasingly recognized as important drivers of local invasion and metastasis. Cortactin, as an actin-binding protein, is closely associated with invadopodia through interacting with proteins. Formin-like 2 (FMNL2), a member of diaphanous-related formins which act as nucleation factors, plays an important role in tumor progression. But whether cortactin can interact with FMNL2 to promote invadopodia formation and the role of FMNL2 in invadopodia formation are still unknown. Here we found that cortactin directly bound to FMNL2 and elevated the activities of actin polymerization and recycling endosome motility. FMNL2 was necessary for invadopodia formation and function in CRC cells. The interaction of cortactin and FMNL2 could further promote the invadopodia formation and matrix degradation. The stimulation of EGF/cdc42 enhanced the combination of cortactin and FMNL2 to intensify the numbers of invadopodia and the degrees of matrix degradation. In vivo, induction of invadopodia formation via cortactin is essential for the ability of FMNL2 to promote CRC metastasis. Furthermore, up-regulations of FMNL2 and cortactin were highly linked in CRC tissues. Collectively, our work demonstrates a brand-new mechanism of cortacin and FMNL2 at invadopodia in CRC.