C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition

BACKGROUND Skeletal muscle function depends on calcium signaling proteins in the sarcoplasmic reticulum (SR), including the calcium-binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) calcium release channel, and skeletal triadin 95 kDa (trisk95) and junctin, proteins that bind to calsequestrin type 1 (CSQ1) and ryanodine receptor type 1 (RyR1). CSQ1 inhibits RyR1 and communicates store calcium load to RyR1 channels via trisk95 and/or junctin. METHODS In this manuscript, we test predictions that CSQ1's acidic C-terminus contains binding sites for trisk95 and junctin, the major calcium binding domain, and that it determines CSQ1's ability to regulate RyR1 activity. RESULTS Progressive alanine substitution of C-terminal acidic residues of CSQ1 caused a parallel reduction in the calcium binding capacity but did not significantly alter CSQ1's association with trisk95/junctin or influence its inhibition of RyR1 activity. Deletion of the final seven residues in the C-terminus significantly hampered calcium binding, significantly reduced CSQ's association with trisk95/junctin and decreased its inhibition of RyR1. Deletion of the full C-terminus further reduced calcium binding to CSQ1 altered its association with trisk95 and junctin and abolished its inhibition of RyR1. CONCLUSIONS The correlation between the number of residues mutated/deleted and binding of calcium, trisk95, and junctin suggests that binding of each depends on diffuse ionic interactions with several C-terminal residues and that these interactions may be required for CSQ1 to maintain normal muscle function.This work was supported by the Australian Research Council (DP1094219 to AFD and NAB) and a NHMRC Career Development Award (NAB)

Blank Slate: Tactical Placemaking Strategies as a Tool for Neighborhood-Led Activation of Vacant Lots

Master of Landscape ArchitectureDepartment of Landscape Architecture/Regional and Community PlanningMajor Professor Not ListedA common challenge in disinvested urban areas is a high frequency of vacant land, which directly affects the lives of residents in high-vacancy neighborhoods. The increase of crime, induced fear of crime, and creation of visual blight are potential impacts from urban vacancy (Branas et al. 2018; Hadavi et al. 2021). Reactivating vacant land is an important measure in mitigating the negative consequences of urban vacancy and increasing social cohesion of urban neighborhoods. Tactical placemaking offers interim, programmatic solutions that present lowcost, high-impact approaches to instigating neighborhood change through addressing issues related to safety, equity, and disinvestment (Lak & Kheibari 2020, Finn 2014). Maximum effect of tactical placemaking strategies emanates from an understanding of resident preferences and their capacities towards implementation and maintenance. This study examines tactical placemaking strategies as a neighborhood-led approach to activating vacant lots. Eight highvacancy neighborhoods in Eastside Kansas City, Missouri were selected as a case to examine residents’ preferences for tactical placemaking strategies and their interest and ability in implementation and maintenance. First, three vacant lot typologies were identified across the study area: enclosed, post-commercial, and wild. Semi-structured interviews were conducted with twenty-seven residents focusing on the type of vacant lot they were most familiar with. The interviews used a participant-generated photo selection activity to understand preferences towards spaces and activities that could be a strategy to reactivate vacant lots. This activity combined with additional questions captured resident preferences towards the potential reactivation strategies, as well as resident willingness and ability to participate in both creating such spaces and maintaining them. Results of the interviews guided a framework of design recommendations specific to each of the three vacant lot typologies. The recommendations include the necessary resources for project construction within four major criteria including time, money, labor, and expertise, while acknowledging how the strategy is expected to activate the space within three areas of opportunity: skill-building, recreation, and gathering. Each recommendation includes a community development rating to further assist community members with decision-making. While providing residents with the foundation for reactivating and reclaiming underutilized land, this study contributes to the literature regarding tactical placemaking and vacancy typologies. The broader implication of this study is that it offers a practical photo-selection method to learn about people’s preferences for outdoor spaces and activities that could be used in community engagement activities

A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49 ± 0.11-fold and 1.8 ± 0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.This work was supported by the Australian Research Council (DP1094219 to A.F.D. and N.A.B.)

How Thoroughly Do Proposed Nextgen Mid-Term Operational Improvements Address Existing Threats?

The goals of the Federal Aviation Administration’s (FAA) Next Generation Air Transportation System (NextGen) include improved safety, increased capacity, increased efficiency, and reduced environmental impact. The FAA has developed 46 mid-term Operational Improvements (OIs) to facilitate initial realization of these benefits in the 2015 – 2018 timeframe. These OIs describe changes in technologies, policies and procedures from current-day air and ground operations designed to mitigate safety, capacity, efficiency, and environmental issues. The main goal of this project was to investigate how thoroughly threats to safety present in today’s operations are addressed by the OIs. These threats, without mitigation, could remain threats in the mid-term, potentially compromising the intended NextGen safety benefits. To address this concern, we extracted threats to safety from 200 Aviation Safety Reporting System incident reports filed by tower air traffic controllers over a five-year period. We then evaluated whether these threats are addressed by the mid-term OIs

Cardiac ryanodine receptor activation by a high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, bu

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca 2+ -binding protein changes in CSQ2 knockout mice

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n = 8) contained 24 ± 2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430 ± 20 μmol/kg (with SR Ca2+ likely <250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidinerich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation

Most soil trophic guilds increase plant growth: a meta-analytical review

Trophic cascades are important drivers of plant and animal abundances in aquatic and aboveground systems, but in soils trophic cascades have been thought to be of limited importance due to omnivory and other factors. Here we use a meta-analysis of 215 studies with 1526 experiments that measured plant growth responses to additions or removals of soil organisms to test how different soil trophic levels affect plant growth. Consistent with the trophic cascade hypothesis, we found that herbivores and plant pathogens (henceforth pests) decreased plant growth and that predators of pests increased plant growth. The magnitude of this trophic cascade was similar to that reported for aboveground systems. In contrast, we did not find evidence for trophic cascades in decomposer- and symbiont-based (henceforth mutualist) food chains. In these food chains, mutualists increased plant growth and predators of mutualists also increased plant growth, presumably by increasing nutrient cycling rates. Therefore, mutualists, predators of mutualists and predators of pests all increased plant growth. Further, experiments that added multiple organisms from different trophic levels also increased plant growth. As a result, across the dataset, soil organisms increased plant growth 29% and non-pest soil organisms increased plant growth 46%. Omnivory has traditionally been thought to confound soil trophic dynamics, but here we suggest that omnivory allows for a simplified perspective of soil food webs – one in which most soil organisms increase plant growth by preying on pests or increasing nutrient cycling rates. An implication of this perspective is that processes that decrease soil organism abundance (e.g. soil tillage) are likely to decrease aboveground productivity. Synthesis Soil foodwebs have resisted generalizations due to their diversity and interconnectedness. Here we use results from a meta-analysis to inform a simplified perspective of soil foodwebs: one in which most soil trophic guilds increase plant growth. Our review also includes the first widespread support for the presence of trophic cascades in soils

Introduction effort, climate matching and species traits as predictors of global establishment success in non-native reptiles

Non-native reptiles are often detrimental to native communities and ecosystems and can be extremely difficult to manage once established. Thus, there is considerable interest in predicting the likelihood of establishment of nonnative reptiles. We assessed three hypotheses describing possible factors contributing to the successful establishment of introduced reptiles in an effort to better identify potential invaders