Changes In Actin Polymerization Research Articles

Presynaptic plasticity is associated with actin polymerization

Modifications of presynaptic short-term plasticity as a result of actin polymerization were tested in rat hippocampal slices using a paired-pulse paradigm. Paired-pulse stimulation of Schaffer collaterals with 70 ms interpulse interval was continuously presented every 30 s before and during perfusion with jasplakinolide, an activator of actin polymerization. Jasplakinolide application resulted in the potentiation of CA3-CA1 responses accompanied by paired-pulse facilitation decrease, these effects suggesting presynaptic modifications. The jasplakinolide-induced potentiation significantly depended on the initial paired-pulse facilitation values. These data indicate that jasplakinolide-mediated changes in actin polymerization may promote high probability of release. Less typical for CA3-CA1 synapses responses with very low initial paired-pulse facilitation or paired-pulse depression (close to 1 or even lower) demonstrated the potentiation of the second, but not the first amplitude in a pair, paired-pulse ratio significantly increasing from 0.8 to 1.0 in average. This may suggest a negative impact of jasplakinolide on the mechanisms underlying paired-pulse depression. Nonetheless, actin polymerization promotes potentiation, though patterns of this activation may differ depending on initial input characteristics. We conclude that in addition to increased neurotransmitter release probability other mechanisms known to suppress paired pulse facilitation may be also involved in effects of jasplakinolide.

Presynaptic Plasticity Is Associated with Actin Polymerization

Modulation of presynaptic short-term plasticity induced by actin polymerization was studied in rat hippocampal slices using the paired-pulse paradigm. Schaffer collaterals were stimulated with paired pulses with a 70-ms interstimulus interval every 30 s before and during perfusion with jasplakinolide, an activator of actin polymerization. Jasplakinolide application resulted in the increase in the amplitudes of CA3-CA1 responses (potentiation) accompanied by a decrease in the paired-pulse facilitation, suggesting induction of presynaptic modifications. Jasplakinolide-induced potentiation depended on the initial paired-pulse rate. These data indicate that the jasplakinolide-mediated changes in actin polymerization increased the probability of neurotransmitter release. Less typical for CA3-CA1 synapses responses, such as a very low paired-pulse ratio (close to 1 or even lower) or even paired-pulse depression, were affected differently. Thus, jasplakinolide caused potentiation of the second, but not the first response to the paired stimulus, which increased the paired-pulse ratio from 0.8 to 1.0 on average, suggesting a negative impact of jasplakinolide on the mechanisms promoting paired-pulse depression. In general, actin polymerization facilitated potentiation, although the patterns of potentiation differed depending on the initial synapse characteristics. We conclude that in addition to the increase in the neurotransmitter release probability, jasplakinolide induced other actin polymerization-dependent mechanisms, including those involved in the paired-pulse depression.

Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p<0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.

Alteration of twinfilin1 expression underlies opioid withdrawal-induced remodeling of actin cytoskeleton at synapses and formation of aversive memory.

Exposure to drugs of abuse induces alterations of dendritic spine morphology and density that has been proposed to be a cellular basis of long-lasting addictive memory and heavily depend on remodeling of its underlying actin cytoskeleton by the actin cytoskeleton regulators. However, the actin cytoskeleton regulators involved and the specific mechanisms whereby drugs of abuse alter their expression or function are largely unknown. Twinfilin (Twf1) is a highly conserved actin-depolymerizing factor that regulates actin dynamics in organisms from yeast to mammals. Despite abundant expression of Twf1 in mammalian brain, little is known about its importance for brain functions such as experience-dependent synaptic and behavioral plasticity. Here we show that conditioned morphine withdrawal (CMW)-induced synaptic structure and behavior plasticity depends on downregulation of Twf1 in the amygdala of rats. Genetically manipulating Twf1 expression in the amygdala bidirectionally regulates CMW-induced changes in actin polymerization, spine density and behavior. We further demonstrate that downregulation of Twf1 is due to upregulation of miR101a expression via a previously unrecognized mechanism involving CMW-induced increases in miR101a nuclear processing via phosphorylation of MeCP2 at Ser421. Our findings establish the importance of Twf1 in regulating opioid-induced synaptic and behavioral plasticity and demonstrate its value as a potential therapeutic target for the treatment of opioid addiction.

Opioids Cause Vascular Remodeling via Changes in Cofilin‐ERK Signaling: Female Mice Present Higher Risk of Developing Morphine‐induced Cardiovascular Disease than Male Mice

According to the most recent CDC Annual Surveillance Report of Drug Related Risks and Outcomes, more than 47,600 people died from an opioid overdose in 2017 in the United States. The magnitude of the national crisis raises questions on intersecting points between opioid abuse and the main systems of the human body. Recent statistical analyses have shown that chronic use of prescription or illicit opioids leads to an increased risk of a cardiovascular event, which was higher among women than men. We hypothesized that chronic exposure to exogenous opioids causes sex‐specific vascular remodeling via changes in actin polymerization, leading to an increased risk of cardiovascular disease. In a preliminary study to test our hypothesis, we observed that treatment with the exogenous opioid morphine (≥ 24 hours) induced vascular smooth muscle cell proliferation. Acknowledging the emerging roles of cofilins and extracellular signal‐regulated kinases (ERK) in the regulation of cell proliferation by mediating actin dynamics, we investigated the effects of morphine (MOR) on the intracellular levels of both proteins in vasculature. Mesenteric resistance arteries (MRA) from 10 week old wild type C57BL/6NTac male and female mice were extracted (n=4). The MRA of each animal were isolated and divided equally into control and treatment groups. The control and treated samples were incubated in DMEM with 1% penicillin‐streptomycin at 37°C for 24 hours in the absence or presence of MOR (1μM) respectively. We observed that MOR increased phosphorylated/inactivated cofilin 1.6 fold [arbitrary units (AU), control: 0.59 ± 0.14 vs MOR: 0.95 ± 0.10*, *p<0.05] and increased phosphorylated/activated ERK 1.8 fold (AU, control: 0.51 ± 0.11 vs MOR: 0.90 ± 0.13*, *p<0.05) in the MRA from female mice. On the other hand, MOR decreased phosphorylated ERK 1.5 fold (AU, control: 0.91 ± 0.08 vs MOR: 0.59 ± 0.11*, *p<0.05) but had no effect on the levels of phosphorylated cofilin (p>0.05) in the MRA from male mice. Our data suggest that exogenous opioids change actin dynamics in arteries from both male and female mice. Further, this study is the first to demonstrate that prolonged exposure to morphine may increase cardiovascular risk in females when compared to males.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) (NIH: R00GM118885).Levels of phosphorylated cofilin (left) and ERK (right) in MRA from male and female mice after 24 hours of treatment with morphine (MOR, 1μM) or vehicle.Figure 1

IL-1β regulates cell shape and expression of prg4 independent of actin polymerization in superficial zone chondrocytes

Purpose: The superficial zone (SZ) of articular cartilage plays a multifaceted role in joint homeostasis contributing the biomechanical integrity and metabolic homeostasis of the tissue. In the early stages of osteoarthritis (OA), one of the first observable changes is loss of the SZ. Although the pathogenesis of OA is multifactorial, elevated levels of inflammatory cytokines are one contributing factor to disease progression. Interleukin-1 is an inflammatory cytokine that contributes to joint remodeling by regulating the expression of collagenases, aggrecanases, and nitric oxide (NO). Additionally, decreased expression of the chondroprotective molecule PRG4 is observed in SZ chondrocytes (SZC) in vitro and in the SZ of cartilage explant culture with IL-1 treatment. Studies using chondrocytes isolated from full-thickness cartilage observed changes in cellular mechanics with IL-1β treatment including increased filamentous actin and cellular stiffness. Unlike full-thickness chondrocytes, where actin polymerization leads to loss of the chondrogenic phenotype, the polymerized form of actin is important for the maintenance of the SZ phenotype and expression of PRG4. Although a decrease in PRG4 expression has previously been observed with IL-1β treatment in SZC the mechanism has not yet been fully elucidated. The purpose of this study is to determine whether IL-1β regulates PRG4 expression through an actin-dependent mechanism. Methods: The SZ of articular cartilage was dissected from the metacarpal phalangeal joint of calves aged 6-9 months. SZC were isolated by sequential digestion in 0.5% protease for 45 minutes, followed by 0.1% collagenase for 14-17 hours. SZC were seeded in monolayer culture (52,000/cm2) in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours in culture, SZC were treated with IL-1β (10ng/mL) and maintained for 24 hours prior to harvesting for analysis. The effect of IL-1β on cell morphology and the cytoskeleton was determined. Cellular morphology was assessed using Image J analysis software to calculate cell area and circularity. Gene and protein expression was determined by RT-PCR and Western blotting, respectively. The cytoskeleton polymerization status was determined by performing differential triton extraction containing the soluble portion (G-actin and M-tubulin) and insoluble portion (F-actin and P-tubulin). The fractions were separated by SDS-PAGE and the resultant bands were semi-quantified using Image J analysis software to determine the G-/F-actin ratio and M-/P-tubulin ratio. Confocal imaging of the actin cytoskeleton visualizing the G- and F-actin was performed by confocal microscopy. Experiments were performed with SZC from independent cell isolations. Each experiment was repeated 2-4 times; experiments were performed in triplicate, where appropriate. Student’s T-test was used to evaluate the results and significance assigned at p <0.05. Results: To confirm observations previously reported SZC treated with IL-1β were assessed by RT-PCR to determined PRG4 expression levels. PRG4 mRNA levels significantly decreased with treatment at 24 hours (p<0.05; N=4). The effect of IL-1β treatment on cellular morphology and actin organization was determined. Treatment with IL-1β resulted in no significant changes in cell area, but did result in a significant increase in cell circularity (p<0.001; N=2). Visualization of the G- and F-actin by confocal microscopy yielded no observable differences in the actin structure (N=2). To confirm this, the polymerization status of the actin cytoskeleton was determined by calculating the G-/F-actin ratio. Despite changes in cell circularity, no significant changes in the G-/F-actin ratio were observed (p=0.8; N=4). Changes in the microtubule structure can also induce changes in cellular morphology, thus we investigated whether the microtubule polymerization status was altered with IL-1β treatment, and observed no significant difference in the M-/P-tubulin ratio (p=0.3; N=3), suggesting another regulator of cell shape may be modulating PRG4 expression in SZC. Conclusions: The data suggests that the elongated morphology is an important regulator of PRG4 expression. Despite changes in cell circularity and reduced expression of PRG4, no changes in actin polymerization or tubulin polymerization were observed. Although actin polymerization has previously been shown to regulate PRG4 expression, additional regulators of cellular morphology may also be important for the maintenance of the SZ phenotype.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Sperm-oocyte contact induces outside-in signaling via PYK2 activation

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm – oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline – rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm – oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm – binding sites under conditions where sperm – oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm – oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Sequential recruitment of Rab GTPases during early stages of phagocytosis.

The phagocytosis and destruction of pathogens and dead cells by macrophages is important for innate immunity and tissue maintenance. Multiple Rab family GTPases engage effector molecules to coordinate the early stages of phagocytosis, which include rapid changes in actin polymerization, membrane phospholipids, trafficking and the activation of receptors. Defining the spatiotemporal, sequential recruitment of these Rabs is critical for insights into how phagocytosis is initiated and coordinated. Here, we screened GFP-tagged Rabs expressed in fixed and live cells to identify and stratify those recruited to early phagocytic membranes at stages defined by phospholipid transitions. We propose a sequence of Rabs 35, 13, 8a, 8b, 27a, 10, and 31 that precedes and accompanies phagocytic cup closure, followed after closure by recruitment of endosomal Rabs 5a, 5b, 5c, 14, and 11. Reducing the expression of individual Rabs by siRNA knockdown, notably Rabs 35 and 13, disrupts phagocytosis prior to phagocytic cup closure, confirming a known role for Rab35 and revealing anew the involvement of Rab13. The results enhance our understanding of innate immune responses in macrophages by revealing the sequence of Rabs that initiates phagocytosis.

Effect of cooling (4 °C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in buffalo spermatozoa

Semen cryopreservation is broadly utilized as a part of the bovine reproducing industry, a large portion of the spermatozoa does not survive and the majority of those that do survive experience various molecular and physiological changes that influence their fertilizing capacity. The main aim of this study is to determine the effect of cooling (4 °C) and cryopreservation on cytoskeleton actin, tyrosine phosphorylation and quality of buffalo spermatozoa, and to determine the similarity between in vitro capacitation and cryopreservation induced capacitation like changes. To achieve this, Western blot was used to examine the changes in actin expression and protein tyrosine phosphorylation, whereas changes in actin polymerization, localization of actin and protein tyrosine phosphorylation during capacitation and cryopreservation were evaluated by indirect immunofluorescence technique. Localization studies revealed that the actin localized to flagella and acrosome membrane regions and following, capacitation it migrated towards the acrosome region of sperm. Time dependent increase in actin polymerization and protein tyrosine phosphorylation was observed during in vitro capacitation. The cooling phase (4 °C) and cryopreservation processes resulted in the loss/damage of cytoskeleton actin. In addition, we performed the actin polymerization and protein tyrosine phosphorylation in cooled and cryopreserved buffalo spermatozoa. Interestingly, cooling and cryopreservation induces actin polymerization and protein tyrosine phosphorylation, which were similar to in vitro capacitation (cryo-capacitation). These changes showed 1.3 folds reduction in the sperm quality parameters which includes motility, viability and plasma membrane integrity. Furthermore, our findings indicate that cooling and cryopreservation damages the cytoskeleton actin and also induces capacitation like changes such as protein tyrosine phosphorylation and actin polymerization. This could be one of the main reasons for reduced sperm quality and fertility failure of cryopreserved spermatozoa.

Myocardin Family Members Drive Formation of Caveolae.

Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Mechanical Activation of Cells Induces Chromatin Remodeling Preceding MKL Nuclear Transport

For cells to adapt to different tissues and changes in tissue mechanics, they must be able to respond to mechanical cues by changing their gene expression patterns. Biochemical signaling pathways for these responses have been elucidated, and recent evidence points to the involvement of force-induced deformation of the nucleus. However, it is still unclear how physical cues received at the plasma membrane (PM) spatiotemporally integrate to the functional chromatin organization of the cell nucleus. To investigate this issue, we applied mechanical forces through magnetic particles adhered to the PM of single cells and mapped the accompanying changes in actin polymerization, nuclear morphology, chromatin remodeling, and nuclear transport of soluble signaling intermediates using high-resolution fluorescence anisotropy imaging. Using this approach, we show the timescales associated with force-induced polymerization of actin and changes in the F/G actin ratio resulting in nuclear translocation of the G-actin-associated transcriptional cofactor, megakaryoblastic acute leukemia factor-1 (MKL). Further, this method of measuring nuclear organization at high spatiotemporal resolution with simultaneous force application revealed the physical propagation of forces to the nucleus, resulting in changes to chromatin organization, followed by nuclear deformation. We also describe a quantitative model that incorporates active stresses and chemical kinetics to evaluate the observed timescales. Our work suggests that mechanical activation of cells is accompanied by distinct timescales involved in the reorganization of actin and chromatin assembly, followed by translocation of transcription cofactors from the cytoplasm to the nucleus.

Chemerin Peptides Promote Phagocytosis in a ChemR23- and Syk-Dependent Manner

Chemerin peptides represent a recently identified component of the endogenous anti-inflammatory network that act via the G protein-coupled receptor ChemR23. The role of the chemerin peptide/ChemR23 pathway in phagocytosis, the clearance of apoptotic cells (efferocytosis), and the resolution of inflammation is unknown. In this article, we report that low picomolar concentrations of the chemerin peptide chemerin15 (C15) enhance macrophage (MPhi) phagocytosis of microbial particles and apoptotic cells by up to 360% in vitro. These prophagocytic effects of C15 are significantly impaired in ChemR23(-/-) MPhis and are associated with increased actin polymerization and localization of F-actin to the phagocytic cup. Importantly, pharmacological inhibition of Syk activity completely abrogates the prophagocytic activities of C15 and associated changes in actin polymerization and phagocytic cup formation, suggesting that C15 promotes phagocytosis by facilitating phagocytic cup development in a Syk-dependent manner. During peritoneal inflammation, C15 administration (8 pg/mouse) enhances microbial particle clearance and apoptotic neutrophil ingestion by MPhis in wild-type but not ChemR23(-/-) mice, such that levels of apoptotic and necrotic cells at the inflammatory site are profoundly reduced. In contrast, neutralization of endogenous chemerin species during peritoneal inflammation significantly impairs MPhi ingestion of apoptotic neutrophils and zymosan. Our data identify a key role of the chemerin peptide/ChemR23 axis in the efficient clearance of foreign material, efferocytosis, and, hence, the resolution of inflammation. Manipulation of the chemerin peptide/ChemR23 axis may represent a novel therapeutic approach for the treatment of inflammatory pathologies, especially if failure to efficiently clear phagocytic targets has been implicated in their pathogenesis.

Rabbit Muscle Actin Polymerization changes by Ultraviolet –B radiation

AbstractIncrease in Ultraviolet-B flux from the Sun has the potential to alter the protein in mammals. Rabbit muscle was processed and filtered to extract G-actin, which was tested for its protein concentration before exposing to UV-B. It was found that polymerization of actin does get affected exposure of actin monomers to UV-B radiation. Inside the cell, there are many actin binding proteins which might be playing crucial role in G-actin to F-actin formation.

Investigators' Workshop Poster Session 11:30 a.m.-1:30 p.m.

Abstract Lin Xu 1 , L. Zeng 1 , N. R. Rensing 1 , P. Sinatra 1 , S. M. Rothman 1 and M. Wong 1 ( 1 Neurology, Washington University School of Medicine, Saint Louis, MO ) Rationale: Seizures may cause brain injury via a variety of mechanisms, potentially contributing to cognitive deficits in epilepsy patients. Although seizures induce neuronal death in some situations, they may also have “non-lethal” pathophysiological effects on neuronal structure and function, such as modifying dendritic morphology. Previous studies involving conventional fixed tissue analysis have demonstrated a chronic loss of dendritic spines following seizures in animal models and human tissue. More recently, in vivo time-lapse imaging methods have been used to monitor acute changes in spines directly during seizures, but documented spine loss only under severe conditions. Here, we examined effects of secondary generalized seizures induced by kainate, on dendritic structure of neocortical neurons utilizing multiphoton imaging in live mice in vivo and investigated molecular mechanisms mediating these structural changes. Methods: Multiphoton imaging was performed through a craniotomy window to visualize dendrites and associated spines of neocortical neurons in GFP-expressing transgenic mice before and after kainate-induced seizures of varying severity. The same dendrities were followed sequentially by time-lapse imaging, monitoring for overt morphological changes (beading) and changes in spine number over a several hour period. Phalloidin-rhodamine labeling and Western blotting were used to analyze changes in actin polymerization and actin-regulatory factors. Results: Higher stage kainate-induced seizures caused dramatic dendritic beading and loss of spines within minutes, in the absence of neuronal death or changes in systemic oxygenation. Although the dendritic beading improved rapidly following the seizures, the spine loss recovered only partially over a several hour period. Kainate seizures also resulted in activation of the actin-depolymerizing factor, cofilin, and a corresponding decrease in filamentous actin, indicating that depolymerization of actin may mediate the morphological dendritic changes. Finally, an inhibitor of the calcium-dependent phosphatase, calcineurin, antagonized the effects of seizures on cofilin activation and spine morphology. Conclusions: These dramatic in vivo findings demonstrate that seizures produce acute dendritic injury in neocortical neurons via calcineurin-dependent regulation of the actin cytoskeleton and suggest novel therapeutic targets for preventing seizure-induced brain injury.

Diaphanous 1 and 2 Regulate Smooth Muscle Cell Differentiation by Activating the Myocardin-Related Transcription Factors

We have previously shown that smooth muscle cell (SMC) differentiation marker gene expression is regulated by the small GTPase, RhoA. The objective of the present study was to determine the contributions of the RhoA effectors, diaphanous 1 and 2 (mDia1 and mDia2), to this regulatory mechanism. mDia1 and mDia2 are expressed highly in aortic SMCs and in a number of SMC-containing organs including bladder, lung, and esophagus. Activation of mDia1/2 signaling by RhoA strongly stimulated SMC-specific promoter activity in multiple cell-types including primary aortic SMCs, and stimulated endogenous SM alpha-actin expression in 10T1/2 cells. Expression of a dominant negative Dia1 variant that inhibits both mDia1 and mDia2 significantly decreased SMC-specific transcription in SMCs. The effects of mDia1 and mDia2 required the presence of SRF and the activity of the myocardin transcription factors and were dependent on changes in actin polymerization. Importantly, stimulation of mDia1/2 signaling synergistically enhanced the activities of the myocardin-related transcription factors, MRTF-A and MRTF-B, and this effect was attributable to increased nuclear localization of these factors. These results indicate that RhoA-dependent signaling through mDia1/2 and the MRTFs is important for SMC-specific gene expression in SMCs.

A maturational model for the study of airway smooth muscle adaptation to mechanical oscillation

It has been shown that mechanical stretches imposed on airway smooth muscle (ASM) by deep inspiration reduce the subsequent contractile response of the ASM. This passive maneuver of lengthening and retraction of the muscle is beneficial in normal subjects to counteract bronchospasm. However, it is detrimental to hyperresponsive airways because it triggers further bronchoconstriction. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in ASM adaptability to mechanical oscillation. Healthy immature airways of both human and animal exhibit hyperresponsiveness, but whether the adaptative properties of hyperresponsive airway differ from normal is still unknown. In this article, we review the phenomenon of ASM adaptation to mechanical oscillation and its relevance and implication to airway hyperresponsiveness. We demonstrate that the age-specific expression of ASM adaptation is prominent using an established maturational animal model developed in our laboratory. Our data on immature ASM showed potentiated contractile force shortly after a length oscillation compared with the maximum force generated before oscillation. Several potential mechanisms such as myogenic response, changes in actin polymerization, or changes in the quantity of the cytoskeletal regulatory proteins plectin and vimentin, which may underlie this age-specific force potentiation, are discussed. We suggest a working model of the structure of smooth muscle associated with force transmission, which may help to elucidate the mechanisms responsible for the age-specific expression of smooth muscle adaptation. It is important to study the maturational profile of ASM adaptation as it could contribute to juvenile hyperresponsiveness.

Methylation Signals Outside the Nucleus

Ezh2 is a member of the polycomb group of proteins and functions in the control of development by catalyzing methylation of lysine residues on histone proteins, thereby causing changes in gene expression. However, Ezh2 exists in the cytoplasm as well as in the nucleus, so Su et al. explored whether the enzyme might have functions independent of its known role in modification of chromatin in the nucleus. Ezh2 has been reported to associate with the guanine nucleotide exchange factor Vav1, which is an important component of T cell signaling, particularly changes in actin polymerization in response to stimulation of the T cell receptor (TCR). Studies of mouse peripheral T cells confirmed interaction of Ezh2 with Vav. Furthermore, T cells lacking Ezh-2 had defective TCR-induced actin polymerization and an impaired proliferative response. Similarly, mouse embryo fibroblasts lacking Ezh2 showed decreased actin polymerization in response to fibroblast growth factor and failed to form surface ruffles normally seen in platelet-derived growth factor (PDGF)-treated cells. These effects could be rescued by expression of a modified form of Ezh2 that was not localized to the nucleus, supporting a role for the cytoplasmic protein independent of its nuclear effects. Proliferation in response to PDGF was also reduced in cells lacking Ezh2. The presumed cytoplasmic methylation target of Ezh2 remains unknown but appears to act at a point between TCR activation and activation of the guanosine triphosphatase (GTPase) Cdc42. Vav1, though, appears not to be modified by Ezh2. Together, the findings indicate that posttranslational modification by methylation has key regulatory roles outside the nucleus, with implications for immune responses to the TCR and cancer biology, where increased expression of Ezh2 in cancer cells is associated with increased metastatic capacity. I-h. Su, M.-W. Dobenecker, E. Dickinson, M. Oser, A. Basavaraj, R. Marqueron, A. Viale, D. Reinberg, C. Wulfing, A. Tarakhovsky, Polycomb group protein Ezh2 controls actin polymerization and cell signaling. Cell 121 , 425-436. [Online Journal]

Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers.

Repair of superficial gastric mucosal injury is accomplished by the process of restitution-migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.