Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. ...Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular Ca(2+)i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ PLCβ- inositol 1,4,5-triphosphate receptor IP3R), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in Ca(2+)i induced by bronchoconstrictors, and this lowering of the Ca(2+)i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in Ca(2+)i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via Gβγ to increase Ca(2+)i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in Ca(2+)i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.
Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, ...vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
We study the nature of feedback mechanisms in the 11 CLASH brightest cluster galaxies (BCGs) that exhibit extended ultraviolet and nebular line emission features. We estimate star formation rates ...(SFRs), dust masses, and starburst durations using a Bayesian photometry-fitting technique that accounts for both stellar and dust emission from the UV through far-IR. By comparing these quantities to intracluster medium (ICM) cooling times and freefall times derived from X-ray observations and lensing estimates of the cluster mass distribution, we discover a tight relationship between the BCG SFR and the ICM cooling time to freefall time ratio, , with an upper limit on the intrinsic scatter of 0.15 dex. Furthermore, starburst durations may correlate with ICM cooling times at a radius of , and the two quantities converge upon reaching the gigayear regime. Our results provide a direct observational link between the thermodynamical state of the ICM and the intensity and duration of BCG star formation activity, and appear consistent with a scenario where active galactic nuclei induce condensation of thermally unstable ICM overdensities that fuel long-duration (>1 Gyr) BCG starbursts. This scenario can explain (a) how gas with a low cooling time is depleted without causing a cooling flow and (b) the scaling relationship between SFR and . We also find that the scaling relation between SFR and dust mass in BCGs with SFRs yr−1 is similar to that in star-forming field galaxies; BCGs with large ( yr−1) SFRs have dust masses comparable to extreme starbursts.
The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in ...attentiondeficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or “endocytic brake,” controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.
We present new Atacama Large Millimeter Array observations of the molecular gas and far-infrared continuum around the brightest cluster galaxy (BCG) in the cool-core cluster MACS 1931.8-2635. Our ...observations reveal (1.9 0.3) × 1010 M of molecular gas, on par with the largest known reservoirs of cold gas in a cluster core. We detect CO(1−0), CO(3−2), and CO(4−3) emission from both diffuse and compact molecular gas components that extend from the BCG center out to ∼30 kpc to the northwest, tracing the UV knots and H filaments observed by the Hubble Space Telescope. Due to the lack of morphological symmetry, we hypothesize that the ∼300 km s−1 velocity of the CO in the tail is not due to concurrent uplift by active galactic nucleus (AGN) jets; rather, we may be observing the aftermath of a recent AGN outburst. The CO spectral line energy distribution suggests that molecular gas excitation is influenced by processes related to both star formation and recent AGN feedback. Continuum emission in Bands 6 and 7 arises from dust and is spatially coincident with young stars and nebular emission observed in the UV and optical. We constrain the temperature of several dust clumps to be 10 K, which is too cold to be directly interacting with the surrounding ∼4.8 keV intracluster medium (ICM). The cold dust population extends beyond the observed CO emission and must either be protected from interacting with the ICM or be surrounded by local volumes of ICM that are several keV colder than observed by Chandra.
Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit ...DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME (
obe
ncorporation
ediated by
nzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings.
The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate.
Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides ...that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.
Several coronagraph designs have been proposed over the last two decades to directly image exoplanets. Among these designs, vector vortex coronagraphs provide theoretically perfect starlight ...cancellation along with small inner working angles when deployed on telescopes with unobstructed pupils. However, current and planned space missions and ground-based extremely large telescopes present complex pupil geometries, including large central obscurations caused by secondary mirrors, which prevent vortex coronagraphs from rejecting on-axis sources entirely. Recent solutions combining the vortex phase mask with a ring-apodized pupil have been proposed to circumvent this issue, but provide a limited throughput for vortex charges . We present pupil plane apodizations for charge 2, 4, and 6 vector vortex coronagraphs that compensate for pupil geometries with circularly symmetric central obstructions caused by on-axis secondary mirrors. These apodizations are derived analytically and allow vortex coronagraphs to retain theoretically perfect nulling in the presence of obstructed pupils. For a charge 4 vortex, we design polynomial apodization functions assuming a grayscale apodizing filter that represent a substantial gain in throughput over the ring-apodized vortex coronagraph design, while for a charge 6 vortex, we design polynomial apodized vortex coronagraphs that have total energy throughput for the entire range of central obscuration sizes studied. We propose methods for optimizing apodizations produced with either grayscale apodizing filters or shaped mirrors. We conclude by demonstrating how this design may be combined with apodizations numerically optimized for struts and primary mirror segment gaps to design terrestrial exoplanet imagers for complex pupils.
ABSTRACT Brightest cluster galaxies (BCGs) are usually quiescent, but many exhibit star formation. Here we exploit the opportunity provided by rest-frame UV imaging of galaxy clusters in the Cluster ...Lensing and Supernovae with Hubble (CLASH) Multi-Cycle Treasury Project to reveal the diversity of UV morphologies in BCGs and to compare them with recent simulations of the cool, star-forming gas structures produced by precipitation-driven feedback. All of the CLASH BCGs are detected in the rest-frame UV (280 nm), regardless of their star formation activity, because evolved stellar populations produce a modest amount of UV light that traces the relatively smooth, symmetric, and centrally peaked stellar distribution seen in the near infrared. Ultraviolet morphologies among the BCGs with strong UV excesses exhibit distinctive knots, multiple elongated clumps, and extended filaments of emission that distinctly differ from the smooth profiles of the UV-quiet BCGs. These structures, which are similar to those seen in the few star-forming BCGs observed in the UV at low redshift, are suggestive of bi-polar streams of clumpy star formation, but not of spiral arms or large, kiloparsec-scale disks. Based on the number of streams and lack of culprit companion galaxies, these streams are unlikely to have arisen from multiple collisions with gas-rich galaxies. These star-forming UV structures are morphologically similar to the cold-gas structures produced in simulations of precipitation-driven active galactic nucleus feedback in which jets uplift low-entropy gas to greater altitudes, causing it to condense. Unobscured star formation rates estimated from CLASH UV images using the Kennicutt relation range up to 80 in the most extended and highly structured systems. The circumgalactic gas-entropy threshold for star formation in CLASH BCGs at 0.2-0.5 is indistinguishable from that for clusters at .
A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required ...for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2‐photon imaging, and cell‐specific gene‐modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations SCaOs and asynchronized Ca2+ oscillations ACaOs) that last for hours. RyRs, TMEM16A, L‐type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.
In this study, we found internal anal sphincter smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations SCaOs and asynchronized Ca2+ oscillations ACaOs). RyRs, TMEM16A, L‐type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter of defecation reflex, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our study provides insight into fecal continence and normal defecation.
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