Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

Attack of the PCR clones: Rates of clonality have little effect on RAD‐seq genotype calls

Interpretation of high‐throughput sequence data requires an understanding of how decisions made during bioinformatic data processing can influence results. One source of bias that is often cited is PCR clones (or PCR duplicates). PCR clones are common in restriction site‐associated sequencing (RAD‐seq) data sets, which are increasingly being used for molecular ecology. To determine the influence PCR clones and the bioinformatic handling of clones have on genotyping, we evaluate four RAD‐seq data sets. Data sets were compared before and after clones were removed to estimate the number of clones present in RAD‐seq data, quantify how often the presence of clones in a data set causes genotype calls to change compared to when clones were removed, investigate the mechanisms that lead to genotype call changes and test whether clones bias heterozygosity estimates. Our RAD‐seq data sets contained 30%–60% PCR clones, but 95% of RAD‐tags had five or fewer clones. Relatively few genotypes changed once clones were removed (5%–10%), and the vast majority of these changes (98%) were associated with genotypes switching from a called to no‐call state or vice versa. PCR clones had a larger influence on genotype calls in individuals with low read depth but appeared to influence genotype calls at all loci similarly. Removal of PCR clones reduced the number of called genotypes by 2% but had almost no influence on estimates of heterozygosity. As such, while steps should be taken to limit PCR clones during library preparation, PCR clones are likely not a substantial source of bias for most RAD‐seq studies.     

Genetic diversity and reproductive mode in two non-native hydromedusae, Maeotias marginata and Moerisia sp., in the upper San Francisco Estuary, California

Reproductive strategy can play a significant role in invasion success and spread. Asexual and sexual reproduction may confer different advantages and disadvantages to a founding population, resulting in varying impacts on genetic diversity and the ability to invade. We investigate the role of reproductive mode in two species of non-native hydromedusae (Maeotias marginata and Moerisia sp.) in the San Francisco Estuary (SFE). Both species can reproduce asexually and sexually. We employed 7?C8 microsatellite markers to determine overall genetic diversity and to investigate contributions of asexual and sexual reproduction to the populations. We found both species had high levels of genetic diversity (Average H_E?=?0.63 and 0.58, Number individuals sampled?=?111 and 277, for M. marginata and Moerisia sp. respectively) but also detected multiple individuals in clonal lineages. We identified the same clones across sampling locations and time, and the index of asexual reproduction (R) was 0.89 for M. marginata and 0.91 for Moerisia sp. Our results suggest both species maintain high population genetic diversity through sexual reproduction, in combination with asexual reproduction, which allows rapid propagation. In addition, we conducted genetic sequence analyses at the ribosomal ITS1 marker, using samples of Moerisia sp. from the SFE and M. lyonsi from Chesapeake Bay. We found 100?% sequence similarity showing that Moerisia sp. in the SFE and Chesapeake Bay are the same species. The two hydromedusae studied here possess the means to propagate rapidly and have high genetic diversity, both of which may allow them to successfully adapt to changing environments and expand their invasions.     

Adverse Effects of Ball-Chain Radio-Collars on Female Mantled Howlers (<Emphasis Type="Italic">Alouatta palliata</Emphasis>) in Panama

The placement of radio- and GPS-collars on primates has advanced the fields of primate behavior and ecology and yielded considerable conservation gains. However, to ensure that the placement of tracking collars remains a humane practice, the reporting of both positive and negative outcomes is required. Here, we evaluate the impacts of radio-collars on the reproduction, behavior, and health of female mantled howlers (Alouatta palliata) on Barro Colorado Island, Panama. In June 2005, as part of a behavioral study, we fitted 16 female howlers with ball-chain radio-collars weighing 1.2% of the mean adult female weight. We monitored these 16 collared females and 64 noncollared females across an annual cycle, and recaptured the collared females in July 2006 for collar removal. The mortality rate for collared females across the study year was 6.3% (N=1), while the net loss of noncollared females from study groups was 15.6% (N=10). All collared females reproduced. The behaviors of collared and noncollared females were not significantly different. However, on recapture to remove all remaining collars (N = 13), 38% of recaptured collared females presented with damage to the dermal layer on the back of the neck and 31% presented with more severe damage extending into the subcutaneous tissue and muscle. Given this physical damage, which was not apparent through binoculars, we cannot recommend using ball-chain radio-collars on female mantled howlers without careful welfare monitoring.     

Evaluating Wetland Restoration Success Using Aquatic Macroinvertebrate Assemblages in the Sacramento Valley,California

Currently, there is little professional consensus as to which ecological metrics should be used to measure restoration success in wetlands. Aquatic macroinvertebrate communities have many qualities to recommend them as useful metrics in this manner; yet, they have not been widely used to evaluate wetland restoration success. We examined the macroinvertebrate communities of four restored seasonal wetlands across a chronosequence of postrestoration age and compared them to a remnant natural wetland in the Central Valley of California. We examined two qualitatively different sets of aquatic macroinvertebrate metrics, general measures of community properties (abundance, richness, and diversity) and specific assemblage membership (nonmetric multidimensional scaling and permutational multivariate analysis of variance). Our results using these two different sets of metrics give us different answers. The general measures suggest that wetland macroinvertebrate communities converge on relatively stable values sometime after 10 years postrestoration. The specific assemblage results imply that the particular set of taxa found in restored wetlands is not predictable over the chronosequence we examined. Taken together, our results suggest that aquatic macroinvertebrate communities may be useful for measuring some aspects of restoration success but that there is unlikely to be a final aquatic community pattern indicating restoration success.     

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient''s pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.Download video file.^{(50M, mov)}     

Novel function of cardiac protein kinase D1 as a dynamic regulator of Ca2+ sensitivity of contraction

Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.     

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.     

Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps

The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10 ? resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions.