Actin Depolymerization Research Articles

Grp94 Regulates the Recruitment of Aneural AChR Clusters for the Assembly of Postsynaptic Specializations by Modulating ADF/Cofilin Activity and Turnover.

Temperature is a physiological factor that affects neuronal growth and synaptic homeostasis at the invertebrate neuromuscular junctions (NMJs); however, whether temperature stress could also regulate the structure and function of the vertebrate NMJs remains unclear. In this study, we use Xenopus laevis primary cultures as a vertebrate model system for investigating the involvement of heat shock protein 90 (HSP90) family of stress proteins in NMJ development. First, cold temperature treatment or HSP90 inhibition attenuates the formation of aneural acetylcholine receptor (AChR) clusters, but increases their stability after they are formed, in cultured muscles. HSP90 inhibition specifically affects the stability of aneural AChR clusters and their associated intracellular scaffolding protein rapsyn, instead of causing a global change in cell metabolism and protein expression in Xenopus muscle cultures. Upon synaptogenic stimulation, a specific HSP90 family member, glucose-regulated protein 94 (Grp94), modulates the phosphorylation and dynamic turnover of actin depolymerizing factor (ADF)/cofilin at aneural AChR clusters, leading to the recruitment of AChR molecules from aneural clusters to the assembly of agrin-induced postsynaptic specializations. Finally, postsynaptic Grp94 knock-down significantly inhibits nerve-induced AChR clustering and postsynaptic activity in nerve-muscle co-cultures as demonstrated by live-cell imaging and electrophysiological recording, respectively. Collectively, this study suggests that temperature-dependent alteration in Grp94 expression and activity inhibits the assembly of postsynaptic specializations through modulating ADF/cofilin phosphorylation and activity at aneural AChR clusters, which prevents AChR molecules from being recruited to the postsynaptic sites via actin-dependent vesicular trafficking, at developing vertebrate NMJs.

Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

Human NK Cell Cytoskeletal Dynamics and Cytotoxicity Are Regulated by LIM Kinase.

NK cells provide immune surveillance and host protection against viruses and tumors through their cytotoxic effector function. Cytoskeletal rearrangement is necessary for NK cell lytic granule trafficking and immune synapse formation to trigger apoptosis of targeted cells. LIM kinase (LIMK) regulates F-actin remodeling by phosphorylating cofilin to inhibit actin severing and depolymerization. In this study, in human NK cells, the glucocorticoid dexamethasone downregulated LIMK expression, F-actin accumulation at the immune synapse, lytic granule trafficking, and cytotoxicity. In contrast, the specialized proresolving mediator lipoxin A4 promoted NK cell LIMK expression, lytic granule polarization to the immune synapse and cytotoxicity. Using a LIMK inhibitor, we show that LIMK activity is necessary for NK cell cytotoxicity, including lipoxin A4's proresolving actions. Together, our findings identify LIMK as an important control mechanism for NK cell cytoskeletal rearrangement that is differentially regulated by glucocorticoids and specialized proresolving mediators to influence NK cell cytotoxicity.

The BAR domain of the Arf GTPase-activating protein ASAP1 directly binds actin filaments

The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.

Coronin 2B regulates dendrite outgrowth by modulating actin dynamics.

Cytoskeletal remodeling is indispensable for the development and maintenance of neuronal structures and functions. However, the molecular machinery that controls the balance between actin polymerization and depolymerization during these processes is incompletely understood. Here, we report that coronin 2B, a conserved actin-binding protein, is concentrated at the tips of developing dendrites and that knockdown of coronin 2B inhibits the growth of dendrites. Importantly, coronin 2B interacts with actin and reduces the F-actin/G-actin ratio. Furthermore, the coiled-coil domain of coronin 2B is required for its oligomerization, thus confining coronin 2B to neurite tips. Our findings collectively suggest that coronin 2B is important for promoting dendrite outgrowth by limiting the speed of actin polymerization at growth cones.

Identification and Functional Annotation of Genes Related to Horses' Performance: From GWAS to Post-GWAS.

Simple SummaryIt is assumed that the athletic performance of horses is influenced by a large number of genes; however, to date, not many genomic studies have been performed to identify candidate genes. In this study we performed a systematic review of genome-wide association studies followed by functional analyses aiming to identify the most candidate genes for horse performance. We were successful in identifying 669 candidate genes, from which we built biological process networks. Regulatory elements (transcription factors, TFs) of these genes were identified and used to build a gene–TF network. Genes and TFs presented in this study are suggested to play a role in the studied traits through biological processes related with exercise performance, for example, positive regulation of glucose metabolism, regulation of vascular endothelial growth factor production, skeletal system development, cellular response to fatty acids and cellular response to lipids. In general, this study may provide insights into the genetic architecture underlying horse performance in different breeds around the world.Integration of genomic data with gene network analysis can be a relevant strategy for unraveling genetic mechanisms. It can be used to explore shared biological processes between genes, as well as highlighting transcription factors (TFs) related to phenotypes of interest. Unlike other species, gene–TF network analyses have not yet been well applied to horse traits. We aimed to (1) identify candidate genes associated with horse performance via systematic review, and (2) build biological processes and gene–TF networks from the identified genes aiming to highlight the most candidate genes for horse performance. Our systematic review considered peer-reviewed articles using 20 combinations of keywords. Nine articles were selected and placed into groups for functional analysis via gene networks. A total of 669 candidate genes were identified. From that, gene networks of biological processes from each group were constructed, highlighting processes associated with horse performance (e.g., regulation of systemic arterial blood pressure by vasopressin and regulation of actin polymerization and depolymerization). Transcription factors associated with candidate genes were also identified. Based on their biological processes and evidence from the literature, we identified the main TFs related to horse performance traits, which allowed us to construct a gene–TF network highlighting TFs and the most candidate genes for horse performance.

Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth

SUMMARYSarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in our model. Furthermore, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.

Disrupted actin: a novel player in pathogen attack sensing?

The actin cytoskeleton is widely involved in plant immune responses. The majority of studies show that chemical disruption of the actin cytoskeleton increases plant susceptibility to pathogen infection. Similarly, several pathogens have adopted this as a virulence strategy and produce effectors that affect cytoskeleton integrity. Such effectors either exhibit actin-depolymerizing activity themselves or prevent actin polymerization. Is it thus possible for plants to recognize the actin's status and launch a counterattack? Recently we showed that chemical depolymerization of actin filaments can trigger resistance to further infection via the specific activation of salicylic acid (SA) signalling. This is accompanied by several defence-related, but SA-independent, effects (e.g. callose deposition, gene expression), relying on vesicular trafficking and phospholipid metabolism. These data suggest that the role of actin in plant-pathogen interactions is more complex than previously believed. It raises the question of whether plants have evolved a mechanism of sensing pathological actin disruption that eventually triggers defence responses. If so, what is the molecular basis of it? Otherwise, why does actin depolymerization specifically influence SA content but not any other phytohormone? Here we propose an updated model of actin's role in plant-microbe interactions and suggest some future directions of research to be conducted in this area.

Structural and mechanical remodeling of the cytoskeleton maintains tensional homeostasis in 3D microtissues under acute dynamic stretch

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cell’s environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.

Polymethoxyflavones from Gardenia oudiepe (Rubiaceae) induce cytoskeleton disruption-mediated apoptosis and sensitize BRAF-mutated melanoma cells to chemotherapy

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 μM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 μM vs. >100 μM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs.

The regulatory G protein signaling complex, Gβ5–R7, promotes glucose- and extracellular signal–stimulated insulin secretion

G protein-coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5-R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M-stimulated glucose-stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5-/- islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gβ5-R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5-R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release.

Effects of a-lysophosphatidylinositol (LPI) on kinase activation and cytoskeletal re-arrangements leading to migration in mast cells

Abstract Mast Cells (MCs) initiate allergic responses but also participate in innate inflammatory reactions triggered by infection, tissue damage and cancer. MCs are recruited to affected sites where they release preformed and neo-formed mediators of inflammation. Bioactive lipids (such as sphingosine-1-phosphate, S1P) have demonstrated to induce MC chemotaxis. Recently, α-lysophosphatidilinositol (LPI) has awakened scientific interest due to the discovery of its endogenous receptor, the orphan receptor GPCR55. This receptor has been shown to couple to Gα12/13 pathway, controlling actin polimeryzation and cell migration. In this study, we aimed to characterize the role of LPI in the activation and migration of MCs. Using murine bone marrow-derived MCs, we observed that low concentrations of this lipid (10 nM and 100 nM) induces maximal ERK 1/2 phosphorylation at 15 and 10 minutes, respectively. Also, we found that LIMK (an important G12/13 and Rho-dependent kinase) is phosphorylated after 5 and 10 min after 10 nM or 100 nM LPI, respectively. Both ERK 1/2 and LIMK phosphorylation patterns were reproduced with the selective agonist of GPCR55, O-1602, at equi-potent concentrations to those of the LPI. Using Boyden chamber assays, we observed that LPI provocked higher chemotaxis of MCs when compared with S1P. Finally, using confocal microscopy, we observed that LPI triggered depolymerization of cortical actin, as well as the formation of filopodia between 15 and 30 minutes post stimulation. We concluded that GPR55 expressed in MCs induces important changes on MAPK and LIMK activation and its ligand, LPI, is a potent chemotactic factor of MCs.

Variants in CAPZA2, a member of an F-actin capping complex, cause intellectual disability and developmental delay.

The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. Here, we report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of α and β heterodimer conserved from yeast to human. Vertebrate genomes contain three α subunits encoded by three different genes and CAPZA2 encodes the α2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference; CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, our findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

Lim Kinase1 regulates seizure activity via modulating actin dynamics

Lim kinase1 (LIMK1) plays a vital role in dendritic spine morphogenesis and brain function. However, the mechanism of LIMK1 in epilepsy remains unclear. Our study showed that LIMK1 was upregulated in the hippocampal dentate gyrus (DG) of a pentylenetetrazol (PTZ)-kindled epilepsy rat model. Downregulation of LIMK1 reduced susceptibility to seizures in the PTZ-induced rat model, whereas combined LIMK1 knockdown and jasplakinolide treatment increased the duration of stage 4–5 seizures in PTZ-kindled rats. Via in vitro experiments, we explored the possible mechanism of LIMK1 in seizures. LIMK1 was closely related to actin depolymerization and dendritic spine maturation in Mg2+-free treated hippocampal neurons. Additionally, LIMK1 affected actin polymerization by regulating the level of p-cofilin. Mechanistically, our results show that LIMK1 regulates actin-mediated alterations in dendritic spine morphology in epileptic rats, which requires cofilin phosphorylation. Taken together, our results show that LIMK1 is involved in the spatial control of actin dynamics and kinase signaling in seizures, providing new insights into structural plasticity mechanisms in epilepsy.

Regulation of neutrophil secretion by the lysosomal amino acid transporter, cystinosin

Neutrophil exocytosis is an important immune response, but secretion must be tightly regulated because exacerbated release of the neutrophil toxic mediators is injurious to the host and mediates inflammation. Here we show that neutrophil exocytosis of azurophilic granules is regulated by the cystine lysosomal transporter cystinosin. Cystinosin localized at azurophilic granules and lysosomes in neutrophils. Isolated bone‐marrow‐derived Ctns−/− neutrophils showed exacerbated azurophilic granule secretion under unstimulated conditions, suggesting dysregulation of a cell‐intrinsic mechanism. The defect was specific for azurophilic granules because secretion of gelatinase granules was not affected. Using phalloidin staining, we show that Ctns−/− neutrophils have decreased F‐actin, suggesting disruption of the azurophilic granule‐specific actin barrier. In addition, phalloidin‐based staining showed increased filopodial protrusion formation in Ctns−/− neutrophils, thus affecting cell morphology. Induction of either actin polymerization or depolymerization by jasplakinolide (JK) or cytochalasin, respectively, followed by stimulation with the bacteria‐derived peptide, fMLF, showed that secretion was still significantly increased in Ctns−/− neutrophils, suggesting mechanistic defects beyond impaired actin remodeling. Using Total Internal Reflection Fluorescence Microscope (TIRFM) and Super‐Resolution Radial Fluctuations (SRRF), we show increased numbers of docked granules at the exocytic actin zone in Ctns−/− neutrophils. Immunofluorescence analysis showed increased colocalization of the small GTPase Rab27a with its effector JFC1 in Ctns−/− neutrophils, suggesting that Rab27a drives the increased exocytosis observed in Ctns−/− neutrophils. This was confirmed by treatment with the Rab27a‐JFC1 binding‐specific inhibitor Nexinhib20 (NEI20), which decreased granule docking and impaired secretion of the azurophilic granule marker myeloperoxidase (MPO) in unstimulated and stimulated Ctns−/− neutrophils. Combined treatment with NEI20 and JK further decreased MPO secretion in Ctns−/− neutrophils suggesting the uncoupling of Rab27a activation and actin depolymerization in Ctns−/− neutrophils. This was confirmed by the observation that the number of docking granules decreases in response to the combined treatment. Blocking ERM (Ezrin/Radixin/Moesin) also decreased the number of docked azurophilic granules at the exocytic active zone and impaired MPO secretion in Ctns−/− neutrophils, suggesting the participation of ERM on azurophilic granule exocytosis. In vivo, we found elevated levels of azurophilic granule cargo in plasma from cystinosin‐deficient mice, and Ctns−/− mice showed increased neutrophilic tissue infiltration, denoting systemic activation. Our data suggest that cystinosin is a negative regulator of exocytosis and that neutrophil‐mediated inflammation may play a significant role in disease progression in cystinosis.Support or Funding InformationCRFNIH

Regulator of G protein signaling complex, Gβ5‐R7, promotes glucose‐ and extracellular signal‐stimulated insulin secretion

Glucose‐stimulated insulin secretion (GSIS) is essential for maintaining energy homeostasis. G protein‐coupled receptors (GPCRs) are important modulators of this process by extracellular signals such as hormones, neurotransmitters and non‐glucose metabolites. We investigated the role of Gβ5‐R7, the protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling (RGS) of the R7 family. To analyze the effects of Gnb5 knockout on insulin secretion we used mouse insulinoma MIN6 cell line and pancreatic islets isolated from the Gnb5−/− mice. Consistent with previous work, Gnb5knockout diminished insulin secretion evoked by cholinergic agonist Oxo‐M. Here, we found that Gnb5 knockout also attenuated activity of other GPCR agonists ADP, AVP, GLP‐1, forskolin and, surprisingly, the response to high glucose. Our experiments also provided evidence that Gnb5 knockout eliminated the positive effect of cell adhesion on Oxo‐M‐stimulated GSIS, likely through adhesion GPCR GPR56. Total insulin content and the amount of insulin released in response to K+‐evoked membrane depolarization in Gnb5 −/− islets or MIN6 cells was normal. The pleiotropic effect on several GPCRs led us to the hypothesis that Gβ5‐R7 may play a non‐canonical role in insulin secretory pathway downstream of G protein signaling and glucose‐activated membrane depolarization. We found that Gnb5 knockout does not influence cortical actin depolymerization, but influences activity of protein kinase C, and thus far identified 14‐3‐3ɛ as one of the affected substrates. Together, these results allow us to propose that the Gβ5‐R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway, downstream of G protein signaling and actin depolymerization, but upstream of insulin granule release.Support or Funding InformationThis work was supported by National Institutes of Health Grants R01DK1054127(to V.S.), RO1‐DK073716, DK084236 (to E.B.M), EY11373 (to J.C.).

Commensal microbiota are essential for vascular contractility mediated by actin polymerization

IntroductionCommensal microbiota within a holobiont contribute to the overall health of the host via mutualistic symbiosis. Disturbances in such symbiosis are prominently correlated with a variety of diseases affecting humans, including cardiovascular diseases, which are the number one contributors to mortality. Given that a hallmark of all cardiovascular diseases is changes in vascular function, we hypothesized that depleting microbiota from a holobiont would induce vascular dysfunction.MethodsTo test this hypothesis we used male inbred Sprague Dawley (SD) rats that were either germ‐free (GF) or re‐conventionalized germ‐free (GFC) rat (7 weeks‐old). Re‐conventionalization of GF rats were performed by co‐housing GF rats with conventionally‐raised SD rats for 10 days (1:1 ratio); this allows GF rats to receive a natural, continuous exposure to gut microbiota via coprophagy. Mesenteric resistance arteries were used to evaluate vascular function using a wire myograph. Vascular smooth muscle cells (VSMC) were cultured from aorta to evaluate cell migration via scratch assay; proliferation via IncuCyte; and cofilin phosphorylation (inactive form) via Western blot. We quantified cardiac hypertrophy by dividing the left ventricle weight by 26.73 + tibia length3. (Statistics: t‐test: p<0.05: *vs. GF, n=5–6/group)ResultsGF rats had a lower LV mass compared to GFC (GF: 6.9±0.2; GFC: 7.5±0.2*). Intriguingly, GF rats presented a reduction in both KCl‐and phenylephrine‐induced contraction and GFC ameliorated these responses [KCl: Emax (mN): GF: 4.8±1.0 vs. GFC: 8.5±1.1* and phenylephrine: Emax (mN): GF: 6.9±1.3 vs. GFC: 11.7±0.7*]. Given that no differences were observed in acetylcholine‐induced relaxation, we hypothesized that the hyporesponsiveness of the GF arteries were due to cytoskeleton dynamics. Indeed, we observed that GF rats present a reduction in cofilin phosphorylation (phosphoCofilin/TotalCofilin; AU: GF: 0.3±0.03 vs. GFC: 0.6±0.11*). Decreased phospho‐cofilin leads to its active form and subsequently actin depolymerization in VSMCs.ConclusionThis study shows that commensal bacteria are essential for normal structure and function of the vascular system of the host.Support or Funding InformationNIH: R00GM118885

Alpha-Smooth Muscle Actin-Positive Perivascular Cells in Diabetic Retina and Choroid.

Structural alterations of pericytes in microvessels are important features of diabetic retinopathy. Although capillary pericytes had been known not to have α-smooth muscle actin (αSMA), a recent study revealed that a specific fixation method enabled the visualization of αSMA along retinal capillaries. In this study, we applied snap-fixation in wild type and streptozotocin-induced diabetic mice to evaluate the differences in vascular smooth muscle cells of the retina and the choroid. Mice eyeballs were fixed in ice-cold methanol to prevent the depolymerization of filamentous actin. Snap-fixated retina showed αSMA expression in higher-order branches along the capillaries as well as the arterioles and venules, which were not detected by paraformaldehyde fixation. In contrast, most choriocapillaris, except those close to the arterioles, were not covered with αSMA-positive perivascular mural cells. Large choroidal vessels were covered with more αSMA-positive cells in the snap-fixated eyes. Diabetes induced less coverage of αSMA-positive perivascular mural cells overall, but they reached higher-order branches of the retinal capillaries, which was prominent in the aged mice. More αSMA-positive pericytes were observed in the choroid of diabetic mice, but the αSMA-positive expression reduced with aging. This study suggests the potential role of smooth muscle cells in the pathogenesis of age-related diabetic retinopathy and choroidopathy.

Villin controls the formation and enlargement of punctate actin foci in pollen tubes.

Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes.