Enhanced Actin Polymerization Research Articles

Solo regulates the localization and activity of PDZ-RhoGEF for actin cytoskeletal remodeling in response to substrate stiffness.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.

Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

B-308 Focal Adhesion Kinase Activation is Involved in Tension Development During Contractile Stimulation of Mouse Detrusor Smooth Muscle to Modulate Bladder Dysfunction-Related Lower Urinary Tract Symptoms

Abstract Background The function of the bladder is to store and expel urine. Bladder dysfunction-related lower urinary tract symptoms (LUTS) is a common urological problem and negatively affects health-related quality of life. Investigation of detrusor muscle contraction mechanisms is necessary to understand its underlying pathophysiology. Voiding occurs through bladder muscle contraction, which is mediated primarily by the increased intracellular Ca2+ concentration-induced myosin regulatory light chain (MLC) phosphorylation. Recent studies showed that focal adhesion kinase (FAK)-regulated focal adhesion assembly and actin polymerization are involved in vascular and airway smooth muscle contraction. However, the role of FAK activation in visceral smooth muscle contraction is not well documented. This study aims to determine if FAK activation is involved in mouse detrusor smooth muscle contraction. Methods Normal adult male and female mouse bladders were harvested. The trigone and dome areas were cut, and the bladder body was used. The mucosa was carefully removed. Detrusor muscle strips (1.5–2 × 8–10 mm) were prepared and then suspended in a 20 mL tissue bath filled with physiological Krebs solution and bubbled with 95% O2 and 5% CO2 to obtain a pH of 7.4 at 37 °C. The isometric contraction was recorded. After establishing a stable baseline, the strips were stimulated with 120 mM potassium chloride (KCl), which was used to normalize the contractility induced by subsequent stimuli. One of the following specific inhibitors or the same volume of vehicle (DMSO) was applied and incubated for 30 min: PF-573228 (2 µM, a FAK inhibitor) or latrunculin B (1 µM, an inhibitor of actin polymerization). Then, the contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5–10−4.5 M ) were measured. In a separate experiment, the detrusor strips were stimulated with CCh (10 µM) after incubation with PF-573228 (2 µM) or vehicle (DMSO) for 30 min. Once the peak contraction was reached, the strips were snap-frozen for immunoblotting analysis of the phosphorylated FAK (p-FAK) and MLC (p-MLC). Statistical analysis was performed (GraphPad Prism 6). The contractile responses to KCl and EFS between vehicle and inhibitor groups were compared using a paired t-test. Nonlinear regression was used to compare CCh-induced dose-response curves. For the expression of phosphorylated proteins, one-way ANOVA, followed by Tukey’s posthoc test, was used for analysis. Results KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle group (P < 0.0001). The contractile responses induced by EFS were significantly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (P < 0.05) or latrunculin B at 16 and 32 Hz (P < 0.01). Following the application of PF-573228 or latrunculin B, CCh-induced dose-response contractions were significantly lower than those in the corresponding vehicle-treated strips (P = 0.0021 and 0.0003, respectively). Immunoblotting analysis demonstrated that CCh stimulation caused increased expression of p-FAK and p-MLC, whereas preincubation with PF-573228 led to decreased expression of p-FAK but not p-MLC Conclusion FAK activation is involved in tension development induced by contractile stimulation in mouse detrusor muscle, probably through enhancing actin polymerization, but not MLC phosphorylation.

Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells.

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion.

Actin crosslinking by α-actinin averts viscous dissipation of myosin force transmission in stress fibers

Contractile force generated in actomyosin stress fibers (SFs) is transmitted along SFs to the extracellular matrix (ECM), which contributes to cell migration and sensing of ECM rigidity. In this study, we show that efficient force transmission along SFs relies on actin crosslinking by α-actinin. Upon reduction of α-actinin-mediated crosslinks, the myosin II activity induced flows of actin filaments and myosin II along SFs, leading to a decrease in traction force exertion to ECM. The fluidized SFs maintained their cable integrity probably through enhanced actin polymerization throughout SFs. A computational modeling analysis suggested that lowering the density of actin crosslinks caused viscous slippage of actin filaments in SFs and, thereby, dissipated myosin-generated force transmitting along SFs. As a cellular scale outcome, α-actinin depletion attenuated the ECM-rigidity-dependent difference in cell migration speed, which suggested that α-actinin-modulated SF mechanics is involved in the cellular response to ECM rigidity.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network

BackgroundExcessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear.MethodsLC–MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 μm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC–MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype.ResultsLysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis.ConclusionsLH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

Stress Elicits Contrasting Effects on Rac1-Cofilin Signaling in the Hippocampus and Amygdala.

There is accumulating evidence for contrasting patterns of stress-induced morphological and physiological plasticity in glutamatergic synapses of the hippocampus and amygdala. The same chronic stress that leads to the formation of dendritic spines in the basolateral amygdala (BLA) of rats, leads to a loss of spines in the hippocampus. However, the molecular underpinnings of these divergent effects of stress on dendritic spines are not well understood. Since the activity of the Rho GTPase Rac1 and the actin-depolymerizing factor cofilin are known to play a pivotal role in spine morphogenesis, we investigated if alterations in this signaling pathway reflect the differential effects of stress on spine plasticity in the hippocampus and amygdala. A day after the end of chronic immobilization stress (2 h/day for 10 days), we found a reduction in the activity of Rac1, as well as its effector p21-activated kinase 1 (PAK1), in the rat hippocampus. These changes, in turn, decreased cofilin phosphorylation alongside a reduction in the levels of profilin isoforms. In striking contrast, the same chronic stress increased Rac1, PAK1 activity, cofilin phosphorylation, and profilin levels in the BLA, which is consistent with enhanced actin polymerization leading to spinogenesis in the BLA. In the hippocampus, on the other hand, the same stress caused the opposite changes, the functional consequences of which would be actin depolymerization leading to the elimination of spines. Together, these findings reveal a role for brain-region specific differences in the dysregulation of Rac1-to-cofilin signaling in the effects of repeated stress on two brain areas that are implicated in the emotional and cognitive symptoms of stress-related psychiatric disorders.

Delineating the mechanistic pathway and therapeutic potential of Siglec-6 in Chronic Lymphocytic Leukemia

Abstract Sialic acid-binding immunoglobulin-like lectins (Siglec) regulate immune function via glycan recognition. Here, we show for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in chronic lymphocytic leukemia (CLL). Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ CLL− bone marrow stromal cells. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a Siglec-6+ CLL mouse model (Siglec-6 x TCL1). Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating in vivo efficacy of Siglec-6/CD3 targeted bispecific antibody. Natarajan Muthusamy: NIH-R21 Grant and Pelotonia Idea grant Jessica Nunes: Pelotonia Graduate Fellowship

The Fer tyrosine kinase protects sperm from spontaneous acrosome reaction

The physiological acrosome reaction occurs after mammalian spermatozoa undergo a process called capacitation in the female reproductive tract. Only acrosome reacted spermatozoon can penetrate the egg zona-pellucida and fertilize the egg. Sperm also contain several mechanisms that protect it from undergoing spontaneous acrosome reaction (sAR), a process that can occur in sperm before reaching proximity to the egg and that abrogates fertilization. We previously showed that calmodulin-kinase II (CaMKII) and phospholipase D (PLD) are involved in preventing sAR through two distinct pathways that enhance F-actin formation during capacitation. Here, we describe a novel additional pathway involving the tyrosine kinase Fer in a mechanism that also prevents sAR by enhancing actin polymerization during sperm capacitation. We further show that protein-kinase A (PKA) and the tyrosine-kinase Src, as well as PLD, direct Fer phosphorylation/activation. Activated Fer inhibits the Ser/Thr phosphatase PP1, thereby leading to CaMKII activation, actin polymerization, and sAR inhibition.

Truncation of CXCL8 to CXCL8(9-77) enhances actin polymerization and in vivo migration of neutrophils.

CXCL8 is the principal human neutrophil-attracting chemokine and a major mediator of inflammation. The chemokine exerts its neutrophil-chemotactic and neutrophil-activating activities via interaction with glycosaminoglycans (GAGs) and activation of the G protein-coupled receptors (GPCRs) CXCR1 and CXCR2. Natural CXCL8 displays an exceptional degree of amino (NH2 )-terminal heterogeneity. Most CXCL8 forms result from proteolytic processing of authentic CXCL8(1-77). Here, we compared the potencies to activate and recruit neutrophils of the 3 most abundant natural CXCL8 forms: full-length 77 amino acid CXCL8 and the 2 major natural truncated forms lacking 5 or 8 NH2 -terminal amino acids. NH2 -terminal truncation hardly affected the capacity of CXCL8 to induce shedding of CD62L or to up-regulate the expression of the adhesion molecules CD11a, CD11b, or CD15 on human neutrophils. In addition, the potency of CXCL8 to induce neutrophil degranulation and its effect on phagocytosis remained unaltered upon removal of 5 or 8 NH2 -terminal residues. However, NH2 -terminal truncation strongly potentiated CXCL8-induced actin polymerization. CXCL8(6-77) and CXCL8(9-77) showed a comparable capacity to induce Ca2+ signaling in human neutrophils and to direct in vitro neutrophil migration. Strikingly, the ability of CXCL8(9-77) to recruit neutrophils into the peritoneal cavity of mice was significantly enhanced compared to CXCL8(6-77). These results suggest that NH2 -terminal truncation influences specific biological activities of CXCL8 and indicate that CXCL8(9-77) may be the most potent neutrophil-attracting CXCL8 form in vivo.

NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis.

The development of cerebral cortex requires spatially and temporally orchestrated proliferation, migration, and differentiation of neural progenitor cells (NPCs). The molecular mechanisms underlying cortical development are, however, not fully understood. The neural cell adhesion molecule (NCAM) has been suggested to play a role in corticogenesis. Here we show that NCAM is dynamically expressed in the developing cortex. NCAM expression in NPCs is highest in the neurogenic period and declines during the gliogenic period. In mice bearing an NPC-specific NCAM deletion, proliferation of NPCs is reduced, and production of cortical neurons is delayed, while formation of cortical glia is advanced. Mechanistically, NCAM enhances actin polymerization in NPCs by interacting with actin-associated protein profilin2. NCAM-dependent regulation of NPCs is blocked by mutations in the profilin2 binding site. Thus, NCAM plays an essential role in NPC proliferation and fate decision during cortical development by regulating profilin2-dependent actin polymerization.

Gas7 regulates mitochondrial morphology and physiology

Gas7 belongs to the F‐BAR family of proteins and has been shown to be involved in actin and microtubule dynamics. Gas7 is mainly expressed in mouse brain and testicular tissues. In primary cultured neurons, Gas7 enhances actin polymerization and neurite outgrowth. Mutations in PARKIN and PINK1 genes, which play major roles in mitochondrial quality control and mitochondrial autophagy, are often associated with Parkinson's disease. Here, we report a role of Gas7 in mitochondrial distribution, physiological functions, and enhancement of PINK1‐parkin‐mediated mitochondrial autophagy. In the brain of gas7 knockout mice, the mitochondrial aspect ratio is elevated, suggesting a decrease in the level of mitochondrial fragmentation, resulting in an elongated shape. Furthermore, in primary neuronal cultured cells, we found that mitochondrial distribution was restricted around the nucleus, tubular mitochondrial subtypes increased, indicating decreased mitochondrial division, and increased mitochondrial fusion in Gas7 knockout neurons. Gas7 ectopic‐expression in these primary cultured neurons can restore normal morphology and distribution of mitochondria. Overall, our data suggest that Gas7 is required for normal mitochondrial dynamics and therefore may be important for cerebral neurons to maintain mitochondrial function.Support or Funding InformationThis work was supported by an intramural fund from the Institute of Molecular Biology, Academia Sinica and a theme project of Academia Sinica (2323)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PKA and PI3K activities during capacitation protect sperm from undergoing spontaneous acrosome reaction

In order to fertilize the egg, the spermatozoon should undergo a process called acrosomal exocytosis or acrosome reaction (AR), a process which can take place only after a series of biochemical changes collectively called capacitation occur in the female reproductive tract. We present here for the first time the involvement of protein-kinase A (PKA) and phosphatidyl-inositol-3-kinase (PI3K) in protecting sperm from undergoing spontaneous AR (sAR) which decreases fertilization rate. Previously we showed that Calmodulin-kinase II (CaMKII) and phospholipase-D (PLD) prevent the occurrence of sAR in two distinct pathways by enhancing actin polymerization. Here we show that PKA mediates PLD activation, and inhibition of PKA resulting in an increase of sAR and a decrease of F-actin levels, two functions which can be recovered by adding phosphatidic acid (PA), the product of PLD activity. PKA is known to induce CaMKII activation. Inhibition of CaMKII, also enhanced sAR and reduced F-actin levels which can be recovered by adding PA. Inhibition of the PLD pathway resulted in an increase in sAR and a decrease in F-actin levels which can be artificially reversed by inhibition of protein - phosphatase 1 (PP1), and this effect is mediated by PI3K. All together, we showed that PKA via PLD and PI3K activation protects the sperm from undergoing sAR by enhancing actin polymerization.

Zyxin: a mechanotransductor to regulate gene expression.

Cells answer to biochemical, electrical and mechanical signals in the environment, which regulate their behavior. Mechanical signals can propagate through mechanically stiff structures like focal adhesions (FAs). Zyxin, a LIM domain protein, is localized primarily at focal adhesion plaques. Growing evidence suggests that zyxin is a vital mechanotransductor to regulate the gene expression. In this review, we summarize the features of zyxin and the molecular mechanism of how zyxin participate in the cellular activity. An English-language literature search is based on a keyword-based query of multiple databases (MEDLINE, Embase) and bibliographies from identified publications. The references in the selected paper are also considered as an additional source of data. The search was last updated in April 2018; no limitations are applied. Zyxin enhances actin polymerization with the aid of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins in response to mechanical tension, to perform its role in stress fibers (SFs) remodeling and repair. Zyxin can translocate from focal adhesions (FAs) to the nucleus responds to stretch, and regulate gene transcription by interaction with transcription factors like nuclear matrix protein 4 (NMP4). Misregulation of nuclear functions of zyxin appears to be associated with pathogenic effects and diseases, such as prostate cancer and non-small-cell lung cancer. Zyxin is a crucial ingredient of the cellular mechanotransducing system and can modulate the gene expression. Given its clinical relevance, zyxin is also a promising target for the diagnosis and treatment of certain diseases. Understanding the role of zyxin in force sensing and gene expression regulating provides a compelling challenge for future biomechanics studies, and offers attractive evidence for zyxin as a potential diagnostic marker and therapeutic target for clinical diseases.

Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

Actin Corrals G-Protein Coupled Receptors in Ciliary Membrane

Current understanding of the mechanisms underlying G-Protein Coupled Receptor (GPCR) transport within primary cilium is limited and several competing models exist, including motor-driven transport, local binding and free diffusion. Our analysis of the mean square displacement (MSD) of single GPCRs within ciliary membranes as a function of time step magnitude showed predominant mode of transport is diffusion with transient confinement to membrane sub-regions. The mechanism of this confinement is not well understood, and we hypothesize that actin filaments delimit the membrane sub-regions. We performed super-resolution high speed imaging of single GPCRs transfected into murine inner-medullary collecting duct (IMCD3) cells using quantum-dots. Latrunculin A, Cytochalaisin D, and Jasplakinolide was used to alter actin dynamics in primary cilium. The MSD(tau) analysis revealed free and fast local diffusion, followed by confinement to membrane sub-regions, slowing diffusion ∼15 fold. The average diffusion coefficient of the free local diffusion was [2.53 ± 0.42 micro m2 s−1], which is an order of magnitude higher than previous measurements of GPCR diffusion coefficient inside cilium. The average diffusion coefficient of the slow confined diffusion was [0.18 ± 0.03 mm2 s−1]. The size of the corral is calculated to be approximately 260nm. Disruption of actin by Latrunculin-A and Cytochalasin D resulted in increased diffusion coefficient and MSD. Latrunculin A treatment resulted in a 1.53±0.19 fold increase in the diffusion coefficient above vehicle control. Enhancing actin polymerization with Jasplakinolide significantly decreased the diffusion coefficient and MSD(tau). Fast imaging and the MSD analysis following altered actin dynamics demonstrates that the predominant modes of GPCR movement within the ciliary compartment is free local diffusion in highly fluid membrane, followed by transient confinement to membrane sub-regions delimited by actin. Ongoing studies aim to understand the physiological role of the ciliary membrane compartmentalization.

Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia.

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.

EFhd2/swiprosin-1 regulates LPS-induced macrophage recruitment via enhancing actin polymerization and cell migration

Macrophage motility is vital in innate immunity, which contributes strategically to the defensive inflammation process. During bacterial infection, lipopolysaccharide (LPS) potently activates the migration of macrophages via the NF-κB/iNOS/c-Src signaling pathway. However, the downstream region of c-Src that participates in macrophage migration is unclear. EFhd2, a novel actin bundling protein, was evaluated for its role in LPS-stimulated macrophage migration in this study. We found that LPS stimulated the up-regulation, tyrosine phosphorylation and membrane translocation of EFhd2 in macrophages. The absence of EFhd2 inhibited the recruitment of macrophages in the lungs of LPS-induced septic mice. LPS-induced macrophage migration was neutralized by the deletion of EFhd2. EFhd2-mediated up-regulation of NFPs (including Rac1/Cdc42, N-WASP/WAVE2 and Arp2/3 complex) induced by LPS could be used to explain the role of EFhd2 in promoting actin polymerization. Furthermore, the purified EFhd2 could directly promote actin polymerization in vitro. Dasatinib, a c-Src specific inhibitor, inhibited the up-regulation of EFhd2 stimulated by LPS. Therefore, our study demonstrated that EFhd2 might be involved in LPS-stimulated macrophage migration, which provides a potential target for LPS-activated c-Src during macrophage mobilization.