mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.     

Blind Source Separation Techniques for the Decomposition of Multiply Labeled Fluorescence Images

Methods of blind source separation are used in many contexts to separate composite data sets according to their sources. Multiply labeled fluorescence microscopy images represent such sets, in which the sources are the individual labels. Their distributions are the quantities of interest and have to be extracted from the images. This is often challenging, since the recorded emission spectra of fluorescent dyes are environment- and instrument-specific. We have developed a nonnegative matrix factorization (NMF) algorithm to detect and separate spectrally distinct components of multiply labeled fluorescence images. It operates on spectrally resolved images and delivers both the emission spectra of the identified components and images of their abundance. We tested the proposed method using biological samples labeled with up to four spectrally overlapping fluorescent labels. In most cases, NMF accurately decomposed the images into contributions of individual dyes. However, the solutions are not unique when spectra overlap strongly or when images are diffuse in their structure. To arrive at satisfactory results in such cases, we extended NMF to incorporate preexisting qualitative knowledge about spectra and label distributions. We show how data acquired through excitations at two or three different wavelengths can be integrated and that multiple excitations greatly facilitate the decomposition. By allowing reliable decomposition in cases where the spectra of the individual labels are not known or are known only inaccurately, the proposed algorithms greatly extend the range of questions that can be addressed with quantitative microscopy.     

An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.     

A framework for implementing holistic and integrated life cycle sustainability assessment of regional bioeconomy

Purpose

Currently, social, environmental, and economic risks and chances of bioeconomy are becoming increasingly a subject of applied sustainability assessments. Based on life cycle assessment (LCA) methodology, life cycle sustainability assessment (LCSA) aims to combine or integrate social, environmental, and economic assessments. In order to contribute to the current early stage of LCSA development, this study seeks to identify a practical framework for integrated LCSA implementation.

Methods

We select possible indicators from existing suitable LCA and LCSA approaches as well as from the literature, and allocate them to a sustainability concept for holistic and integrated LCSA (HILCSA), based on the Sustainable Development Goals (SDGs). In order to conduct a practical implementation of HILCSA, we choose openLCA, because it offers the best current state and most future potential for application of LCSA. Therefore, not only the capabilities of the software and databases, but also the supported methods of life cycle impact assessments (LCIA) are evaluated regarding the requirements of the indicator set and goal and scope of future case studies.

Results and discussion

This study presents an overview of available indicators and LCIAs for bioeconomy sustainability assessments as well as their link to the SDGs. We provide a practical framework for HILCSA of regional bioeconomy, which includes an indicator set for regional (product and territorial) bioeconomy assessment, applicable with current software and databases, LCIA methods and methods of normalization, weighting, and aggregation. The implementation of HILCSA in openLCA allows an integrative LCSA by conducting all steps in a single framework with harmonized, aggregated, and coherent results. HILCSA is capable of a sustainability assessment in terms of planetary boundaries, provisioning system and societal needs, as well as communication of results to different stakeholders.

Conclusions

Our framework is capable of compensating some deficits of S-LCA, E-LCA, and economic assessments by integration, and shows main advantages compared to additive LCSA. HILCSA is capable of addressing 15 out of 17 SDGs. It addresses open questions and significant problems of LCSAs in terms of goal and scope, LCI, LCIA, and interpretation. Furthermore, HILCSA is the first of its kind actually applicable in an existing software environment. Regional bioeconomy sustainability assessment is bridging scales of global and regional effects and can inform stakeholders comprehensively on various impacts, hotspots, trade-offs, and synergies of regional bioeconomy. However, significant research needs in LCIAs, software, and indicator development remain.

     

Impacts of Channel Reconstruction on Invertebrate Assemblages in a Restored River

Ecosystem restoration often aims to recreate the physical habitat needed to support a particular life‐stage of a focal species. For example, river channel reconstruction, a common restoration practice along the Pacific coast, is typically used to enhance spawning habitat for adult Chinook salmon, a species experiencing large population declines. These restoration efforts rarely consider, however, that altering spawning habitat could have indirect effects on other life‐stages, such as juveniles, which might occur if, e.g. reconstruction alters the benthic food web. To determine how channel reconstruction impacts benthic macroinvertebrates, juvenile Chinook's primary prey, we conducted two studies at a restoration site in the Merced River, California. We asked (1) has gravel enhancement altered invertebrate assemblages in the restored reach compared with an unrestored reach? and, if so, (2) can shifts in the invertebrate community be explained by increased substrate mobility and by reduced heterogeneity that results from restoration? We show that invertebrate abundance and biomass were lower in the restored reach and that these changes were accompanied by a shift from dominance by filter‐feeding caddisflies (Hydropsyche) in the unrestored reach to grazing mayflies (Baetis) in the restored reach. Using an in situ manipulation, we demonstrated that this trend was driven by increased substrate mobility that reduces the abundance of Hydropsyche and by decreased substrate heterogeneity that reduces the abundance of Baetis. Our studies suggest that geomorphic changes typical of reconstructed rivers can alter food webs in ways that may have important implications for supporting the focal species of restoration efforts.     

Analysis of FRET signals in the presence of free donors and acceptors

A method for spectral analysis of Förster resonance energy transfer (FRET) signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Förster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Ef_dp_a, where f_d is the fraction of donor-type molecules participating in donor-acceptor complexes and p_a is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Ef_a. The analysis of intramolecular FRET (e.g., of tandem constructs) yields the product Ep_a. We use our method to determine these values for a tandem construct of cyan fluorescent protein and yellow fluorescent protein and compare them with those obtained by standard acceptor photobleaching and fluorescence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components.     

Piscivore diet response to a collapse in pelagic prey populations

Pelagic fish populations in the upper San Francisco Estuary have experienced significant declines since the turn of the century; a pattern known as the pelagic organism decline (POD). This study investigated food habits of piscivorous fishes over two consecutive fall seasons following the decline of pelagic fish prey. Specifically, this study addressed the contribution of pelagic versus benthic prey to piscivorous fish diets, including the frequency of predation on special-status pelagic species, and the spatial variability in prey consumption. The piscivore community was dominated by Striped Bass and also included small numbers of Sacramento Pikeminnow and Largemouth Bass. Overall, pelagic prey items contributed less than 10% of the diet by weight in both years, whereas pre-POD studies gleaned from the literature found contributions of 39–100%, suggesting a major switch from pelagic to benthic prey resources. Between-year variation in piscivore diets reflected differences in environmental conditions associated with variation in freshwater outflow. No special status fish species were detected in any of the piscivore stomachs examined. The consequences of this pelagic to benthic diet shift warrants further investigation to understand its ecological relevance.     

Quantitative Intensity-Based FRET Approaches—A Comparative Snapshot

Förster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications.     

Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.     

Controls on the Entrainment of Juvenile Chinook Salmon (Oncorhynchus tshawytscha) into Large Water Diversions and Estimates of Population-Level Loss

Diversion of freshwater can cause significant changes in hydrologic dynamics and this can have negative consequences for fish populations. Additionally, fishes can be directly entrained into diversion infrastructure (e.g. canals, reservoirs, pumps) where they may become lost to the population. However, the effect of diversion losses on fish population dynamics remains unclear. We used 15 years of release and recovery data from coded-wire-tagged juvenile Chinook Salmon (Oncorhynchus tshawytscha) to model the physical, hydrological and biological predictors of salvage at two large water diversions in the San Francisco Estuary. Additionally, entrainment rates were combined with estimates of mortality during migration to quantify the proportion of total mortality that could be attributed to diversions. Statistical modeling revealed a strong positive relationship between diversion rate and fish entrainment at both diversions and all release locations. Other significant relationships were specific to the rivers where the fish were released, and the specific diversion facility. Although significant relationships were identified in statistical models, entrainment loss and the mean contribution of entrainment to total migration mortality were low. The greatest entrainment mortality occurred for fish released along routes that passed closest to the diversions and certain runs of Chinook Salmon released in the Sacramento River suffered greater mortality but only at the highest diversion rates observed during the study. These results suggest losses at diversions should be put into a population context in order to best inform effective management of Chinook Salmon populations.