The surface-boundary layer connection across spatial scales of irrigation-driven thermal heterogeneity: An integrated data and modeling study of the LIAISE field campaign

Irrigation in semi-arid regions induces thermal heterogeneity across a range of spatial scales that impacts the partitioning of energy at the surface, the development of the atmospheric boundary layer, and the bidirectional interactions between the atmosphere and the surface. In this analysis, we use data from the Land Surface Interactions with the Atmosphere in the Iberian Semi-Arid Environment (LIAISE) experiment combined with a coupled land–atmosphere model to understand the role of the scales of irrigation-induced, thermal heterogeneity on the surface fluxes and consequently, the development of the diurnal convective boundary layer. The surface heterogeneity is characterized by Bowen ratios that range from ∼0.01 in the irrigated areas to ∼30 in the non-irrigated areas; however, the observed boundary-layers dynamics in both locations are similar. In this analysis, we address the questions of how the surface fluxes impact the development of the boundary-layer dynamics and how the boundary layer influences the diurnal cycle of surface fluxes. To interpret the observations, we introduce a heterogeneity scaling scheme where length scales range from local scale (∼100 m) to regional scale (∼10 km) to investigate the role of scale on surface representation in numerical models and to address the discrepancy between surface observations and their representation in weather and climate models. We find that at the surface, both the available energy and its partitioning depend on spatial scale. The observed boundary-layer properties can be explained through the composite of surface fluxes at the regional scale. Surface fluxes at the local scales are unable to replicate the observed boundary layer — even when including large-scale contributions. We find that non-local boundary layer processes like advection are important for partitioning energy at the local scale. We explore the connection between surface fluxes and the development of the boundary layer and the potential non-local effects on boundary-layer development

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches

Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus

<div><p><u><i>R</i></u>egulator of <u><i>G</i></u> protein <u><i>s</i></u>ignaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF₄¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus.</p></div

Activation of endogenous G proteins with AlF₄¯ induces translocation of endogenous RGS14 from juxtanuclear membranes to cytosolic puncta and the plasma membrane.

<p>Confocal microscopy analysis (A, C, D) and quantification (B) of endogenous RGS14 translocation from the nuclear membrane after activation of endogenous G proteins with AlF₄¯. A significant decrease in endogenous RGS14 localization around the nuclear membrane of B35 cells was observed 10 min after global G protein activation with AlF₄¯. (A) Confocal images of B35 cells incubated with or without (control) AlF₄¯ for indicated times and stained with an anti-RGS14 polyclonal antibody. Boxed regions are enlarged in the insets. (B) Representative confocal image of an untreated (control) B35 cell stained with the RGS14 polyclonal antibody (red) and counterstained with Hoechst DNA dye (blue) (left column). Right column shows the same cell with lines drawn around the nuclear membrane (white) and cytosol (gray) as described in <i>Materials and Methods</i>. Total fluorescence intensity was measured within the ring around the periphery of the nucleus (bounded by the white lines) and a comparable sized area within the cytosol (bounded by the gray lines) using ImageJ software. Scatterplot shows the ratio of nuclear membrane-to-cytosol localization of endogenous RGS14 in B35 cells following treatment with and without 10 min of AlF₄¯ -induced G protein activation. Nuclear membrane-to-cytosol localization of RGS14 was determined by dividing the average fluorescence intensity (total fluorescence/area) around the nuclear membrane (Nuc Mem) by the average fluorescence intensity of a comparable area in the cytosol (Cyt) as described in <i>Materials and Methods</i>. Each point on the scatter plot represents the Nuc Mem/Cyt fluorescence intensity for a single cell immunostained with an RGS14 antibody and counterstained with Hoechst DNA dye to locate nuclei (n = 35 cells for each experimental condition, 3 independent experiments). Horizontal line shows mean Nuc Mem/Cyt intensity ratio. ****<i>P</i><0.0001 (Student <i>t</i>-test). (C) Localization of endogenous RGS14 increased within clusters around the trans-Golgi network (anti-TGN38) and Golgi (anti- GM130) after G protein activation with AlF₄¯ for 10 min, and (D) and at the plasma membrane in some cells after 15 min. Graphs (D) show fluorescence intensity (arbitrary units; a.u.) of RGS14 along the white lines in the above images. Scale bar, 10 μm in all cases.</p

RGS14 polyclonal antibody specifically recognizes endogenous RGS14 in mouse brain and B35 neuroblastoma cells.

<p>(A) The presence of endogenous RGS14 in brain from wild type (WT) and RGS14 knockout (KO) mice, and B35 rat neuroblastoma cells was analyzed by SDS-PAGE and immunoblotting with an RGS14 polyclonal antibody. (B) Equivalent amounts of B35 cell lysate were resolved by SDS–PAGE and transferred to nitrocellulose membranes. Membranes were probed with an RGS14 antibody or RGS14 antibody pre-adsorbed with five-fold excess (ng protein) purified full-length rat RGS14. (C) Confocal images of B35 cells immunostained with an RGS14 antibody, RGS14 antibody pre-adsorbed with five-fold excess purified full-length RGS14, or no primary antibody (<i>secondary only</i>) followed by Alexa 594 secondary antibody (<i>red</i>). Nuclei were counterstained with Hoechst (<i>blue</i>). Scale bar, 10 μm. All images were acquired and processed using identical settings. Cells shown are representative of approximately 600 cells observed from 40 fields of view across three independent experiments.</p

Endogenous RGS14 is enriched as puncta at various cytosolic compartments of B35 cells.

<p>B35 cells were fixed and co-stained with RGS14 polyclonal antibody (<i>red</i>) and one of several organelle markers (<i>green</i>): (A) Rhodamine phalloidin, F-actin; (B) γ-tubulin, centrosomes; (C) endoplasmic reticulum, KDELR; (D) HSP60, mitochondria; (E) lysosomes; (F) Mann-6; EEA1, early endosomes; (G) GM130, Golgi apparatus; (H) α-tubulin, microtubules. Nuclei were counterstained with Hoechst (<i>blue</i>). For B35 cells co-stained with rhodamine phalloidin to label F-actin (A) and antibody against mitochondrial marker HSP60 (D), cells were fixed with 4% paraformaldehyde in 1X PBS and permeabilized in 0.1% Triton-X. B35 cells co-stained with all other organelles were fixed with 4% PFA in PHEM cytoskeleton stabilizing buffer and permeabilized with methanol. Insets represent magnified boxed regions (2x magnification), and are enlarged to the right of the merged image (detail). Scale bar, 10 μm. White arrowheads point to regions of endogenous RGS14 colocalization with mitochondrial marker HSP60 (D) and early endosome marker EEA1 (F). Cells shown are representative of approximately 600 cells observed from 40 fields of view across 3 independent experiments.</p

Endogenous RGS14 localizes to chromatin-rich compartments in close proximity with RNA polymerase II in the nucleus of B35 cells.

<p>Representative 3D-SIM images of the different types of RGS14 distributions observed in B35 cell nuclei. (A, B) B35 cell nuclei counterstained with Hoechst (<i>white/gray</i>) and immunostained with an anti-RGS14 polyclonal antibody (<i>red</i>). (A) Higher magnification images of boxed regions in the XY-projection (<i>top left</i>) are shown to the right. <i>Bottom row</i> shows orthogonal views marked by the dotted white lines in the XY-projection in the indicated planes (1, XZ; 2, YZ). Yellow arrowheads point to an area of enriched RGS14 staining located within a region of high intensity Hoechst-stained chromatin (chromocenter). Cyan arrows indicate enriched RGS14 staining within a DNA-poor intranuclear channel/tubule. Scale bar, 2 μm. (B) Optical mid section (XY-projection) of a B35 cell nucleus with RGS14 puncta enriched at the periphery of a DNA-rich chromocenter at a nuclear invagination. Higher magnification image of boxed region is shown to the right. Scale bar, 2 μm. (C) Optical mid section of a B35 cell nucleus counterstained with Hoechst (<i>gray</i>) and immunostained with an anti-RGS14 polyclonal antibody (<i>red</i>) and mAb H5 (<i>green</i>), which recognizes the 3′ end of active RNA Polymerase II (Ser2P RNA Pol II). Higher magnification image of boxed region is shown to the right. A subpopulation of RGS14 nuclear puncta colocalize with active RNA Polymerase II (Ser2P RNA Pol II) foci (white arrowheads). Scale bar, 2 μm. Orthogonal views YZ (D) and XZ (E) show RGS14 immunostaining wrapping around Ser2P RNA Pol II foci and spanning across a DNA-poor interchromatin compartment to connect two DNA-rich chromocenters. Scale bar, 1 μm. Graphs show fluorescence intensity (arbitrary units; a.u.) for each channel across the dotted white lines in the direction of the arrow in D and E. Cells shown are representative of approximately 75 cells observed across 3 independent experiments.</p

MAP4K3 inhibits Sirtuin-1 to repress the LKB1-AMPK pathway to promote amino acid-dependent activation of the mTORC1 complex.

mTORC1 is the key rheostat controlling the cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here, we examined the process of MAP4K3 regulation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to achieve robust mTORC1 activation. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor sirtuin-1 (SIRT1) and phosphorylates SIRT1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell

The surface-boundary layer connection across spatial scales of irrigation-driven thermal heterogeneity: An integrated data and modeling study of the LIAISE field campaign

Irrigation in semi-arid regions induces thermal heterogeneity across a range of spatial scales that impacts the partitioning of energy at the surface, the development of the atmospheric boundary layer, and the bidirectional interactions between the atmosphere and the surface. In this analysis, we use data from the Land Surface Interactions with the Atmosphere in the Iberian Semi-Arid Environment (LIAISE) experiment combined with a coupled land–atmosphere model to understand the role of the scales of irrigation-induced, thermal heterogeneity on the surface fluxes and consequently, the development of the diurnal convective boundary layer. The surface heterogeneity is characterized by Bowen ratios that range from ∼0.01 in the irrigated areas to ∼30 in the non-irrigated areas; however, the observed boundary-layers dynamics in both locations are similar. In this analysis, we address the questions of how the surface fluxes impact the development of the boundary-layer dynamics and how the boundary layer influences the diurnal cycle of surface fluxes. To interpret the observations, we introduce a heterogeneity scaling scheme where length scales range from local scale (∼100 m) to regional scale (∼10 km) to investigate the role of scale on surface representation in numerical models and to address the discrepancy between surface observations and their representation in weather and climate models. We find that at the surface, both the available energy and its partitioning depend on spatial scale. The observed boundary-layer properties can be explained through the composite of surface fluxes at the regional scale. Surface fluxes at the local scales are unable to replicate the observed boundary layer — even when including large-scale contributions. We find that non-local boundary layer processes like advection are important for partitioning energy at the local scale. We explore the connection between surface fluxes and the development of the boundary layer and the potential non-local effects on boundary-layer development