Challenges in Validating FLOSS Conguration

Part 3: Licensing, Strategies, and PracticesInternational audienceDevelopers invest much effort into validating configuration during startup of free/libre and open source software (FLOSS) applications. Nevertheless, hardly any tools exist to validate configuration files to detect misconfigurations earlier. This paper aims at understanding the challenges to provide better tools for configuration validation. We use mixed methodology: (1) We analyzed 2,683 run-time configuration accesses in the source-code of 16 applications comprising 50 million lines of code. (2) We conducted a questionnaire survey with 162 FLOSS contributors completing the survey. We report our experiences about building up a FLOSS community that tackles the issues by unifying configuration validation with an external configuration access specification. We discovered that information necessary for validation is often missing in the applications and FLOSS developers dislike dependencies on external packages for such validations

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.

Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE' splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca(2+)/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity

CaMKII T305/306 “burst” auto-phosphorylation <i>in vitro</i>.

<p><i>A</i>, The sequence of the CaMKII regulatory domain with T286 in the autoinhibitory region and T305/306 in the CaM-binding region indicated. <i>B</i>, T305/306 phosphorylation was assessed by Western analysis. CaMKII was pre-phosphorylated at T286 on ice; the “burst” was induced by EGTA addition at 30°C or on ice, and stopped after different reaction times. Note the band-shift caused by phosphorylation of additional sites during the “burst” at 30°C, and the basal immuno-detection without phosphorylation reaction. <i>C</i>, Quantification of the relative T305/306 phosphorylation during the “burst” by arbitrary relative immuno-detection values (IDV). Basal immuno-detection prior to the phosphorylation reactions was subtracted in the quantification shown. No increase in phosphorylation was detected on ice. For 30°C, the results indicate an initial phosphorylation rate (at reaction times of 1 min or less) of ∼0.45 min⁻¹ (assuming that saturation represents near-complete phosphorylation; otherwise the rate is even lower). <i>D</i>, The basal immuno-detection with the phospho-T305 antibody prior to the phosphorylation reactions was likely due to background immuno-reactivity, as indicated by comparison to T305/306A mutant CaMKII.</p

Maximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 1 µM Ca²⁺/CaM and measured by the phosphorylation rate of two different substrates (syntide 2 and AC2) in biochemical assays.

<p>Error bars indicate mean ± s.e.m; **: p<0.01, *: p<0.05 in two-tailed t-test. <i>A</i>, CaMKII activity in the different extracts for each kinase form (n = 5 individual assays). <i>B</i>, The average activity of CaMKII from the individual extracts shown separately in panel A (N = 6 different extracts). <i>C</i>, The relative activity of the T286A mutant (compared to wild type activity normalized as 1 for each substrate) based on the results shown in panel B.</p

CaMKII T305 phosphorylation is further reduced for the T286A mutant.

<p><i>A</i>, T305 phosphorylation was detected by Western analysis after reactions corresponding to the kinase activity assays shown in Fig. 1 (1 min at 30°C, but without substrate peptide) or after extended reaction times (10 min). <i>B</i>, Quantification of the relative T305 phosphorylation by the ratio of the phospho-T305 and total CaMKII immuno-detection values (IDV) shows significantly lower phosphorylation of the T286A mutant in our 1 min kinase assay conditions (N = 3 different extracts for each kinase form; *: p<0.05 in two-tailed t-test). The IDV ratio for T286A was ∼28% (±7.6%) of wild type after the 1 min reactions; in the 10 min reaction experiment shown in panel A, this ratio was ∼83%, consistent with the faster wild type reaction reaching saturation faster.</p

Submaximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 0.1 µM Ca²⁺/CaM and measured by the phosphorylation rate of the substrate syntide 2 (in 1 min reactions at 30°C).

<p>Error bars indicate mean ± s.e.m; **: p<0.01, n.s.: p>0.05 in two-tailed t-test. <i>A</i>, T286 autophosphorylation of CaMKII wild type was assessed by Western analysis (left), and quantified by arbitrary relative immuno-detection values (IDV; right). T286 autophosphorylation stimulated by 0.1 µM Ca²⁺/CaM was slower compared to stimulation by 1 µM Ca²⁺/CaM, but the same level of maximal autophosphorylation was still achieved within 1 min reaction time at 30°C. <i>B</i>, Submaximal activation by 0.1 µM Ca²⁺/CaM was verified by comparing one individual preparation of each CaMKII wild type and T286A mutant to the activity induced by 1 µM Ca²⁺/CaM (n = 4 individual assays). <i>C</i>, The average activity of CaMKII from multiple preparations (N = 5) stimulated by 0.1 µM Ca²⁺/CaM did not differ significantly between CaMKII wild type and the T286A mutant. While the ratio of the mean activities of T286A over wild type was similar as observed at maximal stimulation (compare Fig. 1B,C), the variability at submaximal stimulation was greater (with standard deviations of 35–36% of the mean at submaximal stimulation compared to 13–17% at maximal stimulation for syntide 2 substrate).</p

The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms

Calcium-calmodulin-dependent protein kinase II (CaMKII) is well known for its roles in synaptic plasticity. Using a series of molecular replacement experiments, the authors show that the kinase function of CaMKII is required for long-term plasticity and basal AMPA receptor-mediated transmission