Filamentous Actin Content Research Articles

Inhibiting Geranylgeranylation Increases Neurite Branching and Differentially Activates Cofilin in Cell Bodies and Growth Cones

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.

Cell Viscoelasticity as a Function of Substrate Stiffness

While it is known that cells respond to the mechanical properties of their environment, limited information is available regarding mechanosensing and the means by which such stimuli are transduced. The cytoskeleton, a critical component used by cells to sense substrate stiffness, determines the viscoelasticity of cells. Information on cell viscoelasticity as a function of substrate stiffness and vimentin levels can lead to a larger composite model of the mechanism through which cells respond to external mechanical stimuli.3T3 fibroblasts were grown on collagen-coated polyacrylamide gels, of which the stiffness was controlled by varying both the concentrations of acrylamide and bisacrylamide. An atomic force microscope (AFM) was used to perform dynamic micromechanical tests. The force-mapping mode was employed to obtain 24 by 24 maps of force curves. For each force curve, the AFM first indents a cell with a 700 pN force and then applies a 10 Hz sinusoidal strain to the cell. The storage, E', and loss modulus, E'', were calculated by fitting the sinusoidal portion of the applied force and resulting indentation to obtain the amplitude and phase offset.Both E' and E'' increase with substrate stiffness. While the increase in E' is consistent with literature, we found that the ratio of E'' to E' decreases as substrate stiffness increases. This agrees with our hypothesis that cells become more solid-like as the elastic modulus of the extracellular matrix increases. Additionally, the value of E' was higher in vimentin null cells, suggesting that cells change their cytoskeletal composition to adapt to the loss of vimentin. This may involve an increase in the concentration of actin filaments, which contribute more to the elasticity than vimentin. Further experiments will investigate the potential link between vimentin expression and the mechanosensing ability of the cells.

3D Model of Cytokinetic Ring Assembly in Fission Yeast

During fission yeast cytokinesis, actin filaments are nucleated in random directions by cortical Cdc12 and captured by myosin-II motors from neighboring nodes, exerting forces that pull them together into a contractile ring. Contractile ring assembly has been previously described using a stochastic 2D computational model based on a “search, capture, pull and release” mechanism (SCPR). In this model, actin filaments are assumed to polymerize along the cell membrane, consistent with enhanced concentration of actin filaments near the cell membrane in experiments. Recent observations in mutant and wild-type cells have suggested that actin filaments nucleated in the cytoplasm and in non-medial locations can also participate into the assembly of the cytokinetic ring as well as in clump, star, and meshwork actomyosin morphologies. The relative contributions of the non-medially nucleated actin filaments has been debated. In order to examine these effects that involve 3D dynamics and to better understand the mechanisms that confine actin filaments along the cell surface, we extended the SCPR model to 3D. In this model semiflexible actin filaments (represented as beads connected by springs) grow from nodes and they can be captured by myosin in neighboring nodes. Cross-linking by alpha-actinin and fimbrin is represented by an attractive interaction between filament beads. We also implemented cytoplasmic formin nucleation and actin cable incorporation into the contractile ring. We quantified the resulting distribution and orientation of actin around the band of myosin nodes and studied the effects of varying model parameter values. We describe the conditions under which ring, clump, transient star and meshwork structures may form along the cell membrane and compare to prior experiments.

Mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy

To investigate the mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy. Peripheral venous blood was collected at the time of diagnosis and immediately prior to the 4th cycle of an anthracycline-based chemotherapy regimen for patients diagnosed with different stages of breast cancer (n=30), for experimental assays. Blood samples were also collected from a healthy control group (n=17). IL-8 serum concentrations were higher in the patient group than in the control group (p=0.02), and chemotherapy did not further affect this increase. Levels of TNF-α, IL-6, and IL-10 did not differ between controls and patients, or in relation to chemotherapy. Serum levels of nitric oxide (NO) metabolites were elevated following chemotherapy compared to levels detected prior to treatment (p=0.01). When the supernatants of lipopolysaccharide-stimulated mononuclear cells and neutrophils obtained from the patients were assayed for levels of nitrite, these levels were significantly higher and unchanged, respectively, compared with controls. Expression levels of the chemokine receptors, CXCR1 and CXCR2, were significantly reduced in patients compared to controls, and chemotherapy did not further affect these differences. Furthermore, filamentous actin content for IL-8-activated neutrophils was reduced with chemotherapy (median 8.85; range 3.38-13.43) compared to the content detected prior to treatment (median 9.23; range 2.86-22.16) (p=0.001). Elevated systemic levels of IL-8 and NO, desensitization to CXCR activation, and reduction in actin polymerization may affect neutrophil motility in patients before and after chemotherapy.

Spatiotemporal Dynamics of Actomyosin Networks

Rhodamine–phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 kBT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an “order parameter,” peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.

Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling

Inward remodelling of the resistance vasculature is predictive of hypertension and life-threatening cardiovascular events. We hypothesize that the contractile mechanisms responsible for maintaining a reduced diameter over time in response to prolonged stimulation with vasoconstrictor agonists are in part responsible for the initial stages of the remodelling process. Here we investigated the role of vascular smooth muscle (VSM) actin polymerization on agonist-induced vasoconstriction and development of inward remodelling. Experiments were conducted in Sprague-Dawley rat resistance vessels isolated from the cremaster and mesentery. Within blood vessels, actin dynamics of VSM were monitored by confocal microscopy after introduction of fluorescent actin monomers through electroporation and by differential centrifugation to probe globular (G) and filamentous (F) actin content. Results indicated that 4 h of agonist-dependent vasoconstriction induced inward remodelling and caused significant actin polymerization, elevating the F-/total-actin ratio. Inhibition of actin polymerization prevented vessels from maintaining prolonged vasoconstriction and developing inward remodelling. Activation of the small GTPases Rho/Rac/Cdc42 also increased the F-/total-actin ratio and induced inward remodelling, while inhibition of Rho kinase or Rac-1 prevented inward remodelling. Disruption of the actin cytoskeleton reversed the inward remodelling caused by prolonged vasoconstriction, but did not affect the passive diameter of freshly isolated vessels. These results indicate that vasoconstriction-induced inward remodelling is in part caused by the polymerization of actin within VSM cells through activation of small GTPases.

Spatiotemporal Domains in Active Actin Gels

Using a standard actin filament gliding assay, rhodamine-phalloidin labeled actin filaments were visualized moving over a myosin-II coated surface by fluorescence microscopy and filament tracks were analyzed by computer. When micromolar amounts of non-fluorescent phalloidin-stabilised actin were added, filament motion became aligned into a common direction (Butt et al., 2010, J Biol Chem285:4964-4974). Experiments conducted at low filament densities show that, on average, there is a small angular deflection that tends to align the paths of individual colliding filaments. Path alignment is independent of relative track directionality and increases with collision incident angle. Filament elastic deformation during collision events indicates the collision bending energy is ∼20kBT. As increasing amounts of bulk actin are added, individual filament paths become straighter and start to move in a common direction. Spatial correlation analysis shows that increased alignment is due to incremental recruitment of filament tracks into one orientation rather than fusion of seed domains to larger ones. Experiments performed over a wide range of actin filament concentrations and HMM surface densities, show that ordering of filament motion peaks over a range of conditions and decreases at extreme values. This finding differs from theoretical prediction of a critical phase transition (Kraikivski et al., 2006, Phys Rev. Lett.96:258103). Loss of order at high bulk actin concentration might be explained if there is increased filament exchange between bulk, disordered, actin and surface bound material. Loss of order at high myosin surface densities might arise from increased probability of motor attachment that would reduce ability of actin to change its path following a collision. Although our results are broadly compatible with generation of long-range order from mechanics of individual actin filament interactions; other phenomena become important at high protein concentrations.

Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca(2+). Entry of Ca(2+) through the Ca(2+) release-activated Ca(2+) (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient (sgk1(-/-)) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1(-/-) MCs. Confirming previous results, release of β-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1(-/-) MCs compared with sgk1(+/+) MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 μM), MC degranulation was strongly decreased in both sgk1(+/+) and sgk1(-/-) MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1(+/+) MCs and sgk1(-/-) MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1(+/+) MCs but not in sgk1(-/-) MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1(+/+) MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca(2+) entry in mast cells as a novel mechanism regulating actin cytoskeleton.

Notch1 intracellular domain increases cytoplasmic EZH2 levels during early megakaryopoiesis

Notch pathway is a well-known factor in the development of lymphoid lineage. However, its role in the myeloid lineage has remained ambiguous. We looked into the effect of Notch1 on the megakaryocytic lineage commitment and found an increase in megakaryocyte-specific lineage markers upon transfection with Notch1 intracellular domain (NICD). This effect was mediated by Akt whereby constitutive activation of Akt increased the megakaryocyte markers, whereas inhibition of Akt signalling reduced these marker levels. Along with the change in differentiation status, NICD-induced initiation of early megakaryopoiesis was accompanied by an increased cytoplasmic enhancer of zeste homolog-2 (EZH2) expression. This process was found to be Akt-dependent, and inhibition or overexpression of Akt lead to concurrent changes in EZH2 levels. To elucidate the function of EZH2 in the cytoplasm, novel cytoplasmic interactors of EZH2 were identified by co-immunoprecipitation followed by matrix-assisted laser desorption ionization MS/MS-based protein identification, and thus, PDIA1 and LIM domain kinase-1 (LIMK1) were identified. Interaction of EZH2 with LIMK1 changed the activity of cofilin (a downstream target of LIMK1) towards actin filaments, thereby leading to lower filamentous actin content within these cells. Thus, Notch1 not only induces early megakaryopoiesis but also prepares these cells for subsequent morphological changes.

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [(35) S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4(+) CCR4(+) T cells were determined. The basal [(35) S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pK(a) = 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [(35) S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells.

The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell–matrix adhesion

SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell–matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells.

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ∼40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. Multipoint FDAP analysis revealed that G-actin concentration in lamellipodia was comparable to that in the cell body. We also assessed the cellular concentrations of free G-actin, profilin- and thymosin-β4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.

Rho Kinase‐mediated Regulation of Actin Cytoskeleton Dynamics Participates in NFATc3 Nuclear Transport

Chronic hypoxia activates the Nuclear Factor of Activated T-cells isoform c3 (NFATc3) via endothelin-1 (ET-1) and NFATc3 is required for chronic hypoxia-induced pulmonary hypertension. ET-1 elevates Ca2+activating calcineurin which dephosphorylates NFATc3 allowing its nuclear import. RhoA/Rho kinase (ROK) has also been implicated in NFAT activation but the mechanism is unknown. We hypothesized that ET-1 via ROK increases actin cytoskeleton polymerization to mediate NFATc3 nuclear transport. We found that ROK blockade (fasudil) and destabilization of actin cytoskeleton (cytochalasin B) inhibited ET-1-induced increases in NFAT transcriptional activity in isolated pulmonary arteries from NFAT-luciferase reporter mice. The same treatments as well as stabilization of actin cytoskeleton (jasplakinolide) also inhibited ET-1-induced NFATc3-GFP nuclear import in pulmonary arterial smooth muscle cells. None of the drugs affected the increase in cytosolic Ca2+ caused by ET-1 as detected by fura 2. ET-1 also increased filamentous actin content and its co-localization with NFATc3. Both responses were inhibited by fasudil and cytochalasin B. Our results suggest NFATc3 nuclear transport depends on Ca2+ and ROK-mediated actin cytoskeleton polymerization. Funding source: R01 HL088151.

A three-dimensional random network model of the cytoskeleton and its role in mechanotransduction and nucleus deformation

We have developed a three-dimensional random network model of the intracellular actin cytoskeleton and have used it to study the role of the cytoskeleton in mechanotransduction and nucleus deformation. We use the model to predict the deformation of the nucleus when mechanical stresses applied on the plasma membrane are propagated through the random cytoskeletal network to the nucleus membrane. We found that our results agree with previous experiments utilizing micropipette pulling. Therefore, we propose that stress propagation through the random cytoskeletal network can be a mechanism to effect nucleus deformation, without invoking any biochemical signaling activity. Using our model, we also predict how nucleus strain and its relative displacement within the cytosol vary with varying concentrations of actin filaments and actin-binding proteins. We find that nucleus strain varies in a sigmoidal manner with actin filament concentration, while there exists an optimal concentration of actin-binding proteins that maximize nucleus displacement. We provide a theoretical analysis for these nonlinearities in terms of the connectivity of the random cytoskeletal network. Finally, we discuss laser ablation experiments that can be performed to validate these results in order to advance our understanding of the role of the cytoskeleton in mechanotransduction.

Two-dimensional periodic texture of actin filaments formed upon drying.

We found that a solution of actin filaments can form a periodic texture in the process of drying on a flat glass surface in the air; the periodic texture was composed of smooth meandering bundles of actin filaments. We also found that a branched salt crystal grows in the space between the meandering bundles of actin filaments. The distance between the adjacent striae (striation period) in the resulting dried two-dimensional pattern of striation decreased from about 50 to 2 μm, as the ambient temperature was increased from 4 to 40°C at 1 mg/ml actin, and showed an increasing tendency from a few to several tens μm with the increase in the initial concentration of actin filaments from 0.6 to 2.0mg/ml at room temperature. As the speed of drying is increased at a certain temperature, the striation period was also found to decrease. We propose that the formation of the two-dimensional striation pattern of bundles of actin filaments is the result of condensation of proteins due to dehydration, and suggest that the solvent flow from the center to the periphery of the sample causes the meandering of actin filaments.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

β-Actin is a downstream effector of the PI3K/AKT signaling pathway in myeloma cells

Interleukin 6 is the in vivo growth factor of myeloma cells. In response to IL-6 stimulation, the PI3K/AKT signaling pathway is activated in these cells. With comparative proteomic approaches, this study reveals many putative downstream effectors of the PI3K/AKT pathway. Mass spectrometry analysis of excised protein spots from 2-dimensional gel allowed the identification of proteins such as β-Actin, cyclophilin A, E3 SUMO-protein ligase PIAS-NY protein, HSP 27, PML, and transforming growth factor β-2. Among these putative effectors, β-Actin was chosen for further characterization. Phosphorylation of β-Actin by AKT upon IL-6 stimulation was confirmed by western blotting using a phospho-AKT substrate antibody. Interestingly, IL-6 significantly increased cell migration (P < 0.05) and the content of filamentous actin (P < 0.05). Therefore, IL-6 stimulation could have effects on the migration of myeloma cells, and the phosphorylation of β-Actin is probably involved in the process.

Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons

Mice lacking serotonin receptor 1A (Htr1a) display increased anxiety behavior that depends on the expression of the receptor in the forebrain during the third to fifth postnatal weeks. Within the forebrain, Htr1a is prominently expressed in the soma and dendrites of CA1 pyramidal neurons of the hippocampus and these cells undergo rapid dendritic growth and synapse formation during this period. Consistent with a possible role of Htr1a in synaptic maturation, CA1 pyramidal neurons in the knockout mice show increased ramification of oblique dendrites. These findings suggest that Htr1a may shape hippocampal circuits by directly modulating dendritic growth. Here we show that pharmacological blockade of the receptor during the third to fifth postnatal weeks is sufficient to reproduce the increased branching of oblique dendrites seen in knockout mice. Using dissociated hippocampal cultures we demonstrate that serotonin functions through Htr1a to attenuate the motility of dendritic growth cones, reduce their content of filamentous actin and alter their morphology. These findings suggest that serotonin modulates actin cytoskeletal dynamics in hippocampal neurons during a limited developmental period to restrict dendritic growth and achieve a long-term adjustment of neural connectivity.

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium

Cigarette smoke increases the permeability of the lung epithelium. Consequences of increased permeability include increased access of toxins and pathogens from the air spaces to the interstitium and even the blood stream, and leakage of fluids into the air spaces. The mechanisms for permeability alterations have not been elucidated for airway epithelia. By analogy with other types of epithelia, we hypothesized that changes in the phosphorylation status and function of tight junction (TJ) or cytoskeletal proteins might mediate the smoke-induced permeability changes. We investigated the effects of exposure to mainstream cigarette smoke (MS) on cultures of Calu-3 cells, an airway epithelial cell line. Specifically, MS exposure caused increases in phosphorylation of the myosin-binding subunit (MBS) of myosin phosphatase and myosin light chain (MLC), proteins involved in the regulation of actin polymerization. These results implicate activation of Rho kinase (ROCK), consistent with previously reported data indicating that inhibition of ROCK activation suppressed MS-induced increases in permeability. MS exposure also increased polymerized (filamentous) actin (f-actin) content and caused redistribution of the TJ proteins from the normal apical circumferential band to a more basal location. The translocation of the TJ proteins was spatially associated with local increases in both f-actin and macromolecular permeability. Finally, MS exposure increased tyrosine phosphorylation of occludin but not ZO-1 and decreased association between the two TJ proteins. These results indicate that MS exposure causes alterations in cytoskeletal and TJ structure and function, resulting in increased macromolecular permeability that may contribute to the adverse health effects of MS.