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Urban trees are crucial in delivering essential ecosystem services, including air pollution mitigation. This service is influenced by plant associated microbiomes, which can degrade hydrocarbons, support tree health, and influence ecological processes. Yet, our understanding of tree microbiomes remains limited, thus affecting our ability to assess and quantify the ecosystem services provided by trees as complex systems. The main hypothesis of this work was that tree microbiomes concur to hydrocarbon biodegradation, and was tested through three case studies, which collectively investigated two tree micro-habitats (phyllosphere and tree cavity organic soil-TCOS) under various conditions representing diverse ecological scenarios, by applying different culture-based and molecular techniques and at different scales. The integration of all results provided a more comprehensive understanding of the role of microbiomes in urban trees. Firstly, bacterial strains isolated from the phyllosphere of Quercus ilex were characterized, indicating the presence of Plant-Growth Promoting bacteria and strains able to catabolize PAHs, particularly naphthalene and phenanthrene. Secondly, naphthalene biodegradation on artificially spiked Hedera helix leaves was quantified in greenhouse experiments on inoculated and untreated plants. The persistence of the inoculated strain and community structure of epiphytic bacteria were assessed by Illumina sequencing of V5-V6 hypervariable regions of 16S rRNA gene. Results showed that naphthalene degradation was initially faster on inoculated plants but later the degradation rates became similar, probably because bacterial populations with hydrocarbon-degrading abilities gradually developed also on non-inoculated plants. Finally, we explored bacterial and fungal biodiversity hosted by TCOS samples, collected from six large trees located in an urban park and belonging to different species. Microbial communities were characterized by Illumina sequencing of V5-V6 hypervariable regions of bacterial gene 16S rRNA and of fungal ITS1. Results indicated TCOS as a distinct substrate, whose microbiome is determined both by the host tree and by canopy environmental conditions and has a pronounced aerobic hydrocarbon degradation potential. Overall, a better assessment of biodiversity associated with trees and the subsequent provision of ecosystem services constitute a first step toward developing future new microbe-driven sustainable solutions, especially in terms of support for urban green planning and management policy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gandolfi, Canedoli, Rosatelli, Covino, Cappelletti, Sebastiani, Tatangelo, Corengia, Pittino, Padoa-Schioppa, Báez-Matus, Hernández, Seeger, Saati-Santamaría, García-Fraile, López-Mondéjar, Ambrosini, Papacchini and Franzetti.)

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca 2+ -induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca 2+ -induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations., (© 2024 International Society for Neurochemistry.)

Background and Purpose: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated., Experimental Approach: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca 2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca 2+ was examined in PC12 cells transfected with a membrane-tethered Ca 2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca 2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct., Key Results: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca 2+ . A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca 2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca 2+ microdomains inhibited by A740003., Conclusion and Implications: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca 2+ microdomains., (© 2024 British Pharmacological Society.)

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca 2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca 2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca 2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca 2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca 2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion., (© 2020 International Society for Neurochemistry.)

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N -acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela's inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.

Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca 2+ -induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

β-Subunits of the Ca 2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α 1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca 2+ channels (Ca V β2a) and its SH3 domain (β2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with Ca V β2a or β2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca 2+ currents were reduced by Ca V β2a in the presence of exogenous α1-interaction domain. β2a-SH3, but not its dimerization-deficient mutant, also reduced Ca 2+ currents. Na + currents were also diminished following β2a-SH3 injection. Furthermore, β2a-SH3 was still able to reduce Ca 2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both Ca V β2a and β2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics., (© 2019 International Society for Neurochemistry.)

Chromaffin cells of the adrenal gland medulla synthesize and store hormones and peptides, which are released into the blood circulation in response to stress. Among them, adrenaline is critical for the fight-or-flight response. This neurosecretory process is highly regulated and depends on cytosolic [Ca(2+)]. By forming channels at the plasma membrane, pannexin-1 (Panx1) is a protein involved in many physiological and pathological processes amplifying ATP release and/or Ca(2+) signals. Here, we show that Panx1 is expressed in the adrenal gland where it plays a role by regulating the release of catecholamines. In fact, inhibitors of Panx1 channels, such as carbenoxolone (Cbx) and probenecid, reduced the secretory activity induced with the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP, 50 μM) in whole adrenal glands. A similar inhibitory effect was observed in single chromaffin cells using Cbx or (10)Panx1 peptide, another Panx1 channel inhibitors. Given that the secretory response depends on cytosolic [Ca(2+)] and Panx1 channels are permeable to Ca(2+), we studied the possible implication of Panx1 channels in the Ca(2+) signaling occurring during the secretory process. In support of this possibility, Panx1 channel inhibitors significantly reduced the Ca(2+) signals evoked by DMPP in single chromaffin cells. However, the Ca(2+) signals induced by caffeine in the absence of extracellular Ca(2+) was not affected by Panx1 channel inhibitors, suggesting that this mechanism does not involve Ca(2+) release from the endoplasmic reticulum. Conversely, Panx1 inhibitors significantly blocked the DMPP-induce dye uptake, supporting the idea that Panx1 forms functional channels at the plasma membrane. These findings indicate that Panx1 channels participate in the control the Ca(2+) signal that triggers the secretory response of adrenal chromaffin cells. This mechanism could have physiological implications during the response to stress.

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin's ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.