Myosin VI has a One Track Mind Versus Myosin Va When Moving on Actin Bundles or at an Intersection

Myosin VI (myoVI) and myosin Va (myoVa) serve roles both as intracellular cargo transporters and tethers/anchors. In both capacities, these motors bind to and processively travel along the actin cytoskeleton, a network of intersecting actin filaments and bundles that present directional challenges to these motors. Are myoVI and myoVa inherently different in their abilities to interact and maneuver through the complexities of the actin cytoskeleton? Thus, we created an in vitro model system of intersecting actin filaments and individual unipolar (fascin‐actin) or mixed polarity (α‐actinin‐actin) bundles. The stepping dynamics of individual Qdot‐labeled myoVI and myoVa motors were determined on these actin tracks. Interestingly, myoVI prefers to stay on the actin filament it is traveling on, while myoVa switches filaments with higher probability at an intersection or between filaments in a bundle. The structural basis for this maneuverability difference was assessed by expressing a myoVI chimera in which the single myoVI IQ was replaced with the longer, six IQ myoVa lever. The mutant behaved more like myoVI at actin intersections and on bundles, suggesting that a structural element other than the lever arm dictates myoVI's preference to stay on track, which may be critical to its role as an intracellular anchor .     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

Body Mass Index,Muscle Strength and Physical Performance in Older Adults from Eight Cohort Studies: The HALCyon Programme

Objective

To investigate the associations of body mass index (BMI) and grip strength with objective measures of physical performance (chair rise time, walking speed and balance) including an assessment of sex differences and non-linearity.

Methods

Cross-sectional data from eight UK cohort studies (total N = 16 444) participating in the Healthy Ageing across the Life Course (HALCyon) research programme, ranging in age from 50 to 90+ years at the time of physical capability assessment, were used. Regression models were fitted within each study and meta-analysis methods used to pool regression coefficients across studies and to assess the extent of heterogeneity between studies.

Results

Higher BMI was associated with poorer performance on chair rise (N = 10 773), walking speed (N = 9 761) and standing balance (N = 13 921) tests. Higher BMI was associated with stronger grip strength in men only. Stronger grip strength was associated with better performance on all tests with a tendency for the associations to be stronger in women than men; for example, walking speed was higher by 0.43 cm/s (0.14, 0.71) more per kg in women than men. Both BMI and grip strength remained independently related with performance after mutual adjustment, but there was no evidence of effect modification. Both BMI and grip strength exhibited non-linear relations with performance; those in the lowest fifth of grip strength and highest fifth of BMI having particularly poor performance. Findings were similar when waist circumference was examined in place of BMI.

Conclusion

Older men and women with weak muscle strength and high BMI have considerably poorer performance than others and associations were observed even in the youngest cohort (age 53). Although causality cannot be inferred from observational cross-sectional studies, our findings suggest the likely benefit of early assessment and interventions to reduce fat mass and improve muscle strength in the prevention of future functional limitations.     

Motor Domain Phosphorylation Modulates Kinesin-1 Transport

Disruptions in microtubule motor transport are associated with a variety of neurodegenerative diseases. Post-translational modification of the cargo-binding domain of the light and heavy chains of kinesin has been shown to regulate transport, but less is known about how modifications of the motor domain affect transport. Here we report on the effects of phosphorylation of a mammalian kinesin motor domain by the kinase JNK3 at a conserved serine residue (Ser-175 in the B isoform and Ser-176 in the A and C isoforms). Phosphorylation of this residue has been implicated in Huntington disease, but the mechanism by which Ser-175 phosphorylation affects transport is unclear. The ATPase, microtubule-binding affinity, and processivity are unchanged between a phosphomimetic S175D and a nonphosphorylatable S175A construct. However, we find that application of force differentiates between the two. Placement of negative charge at Ser-175, through phosphorylation or mutation, leads to a lower stall force and decreased velocity under a load of 1 piconewton or greater. Sedimentation velocity experiments also show that addition of a negative charge at Ser-175 favors the autoinhibited conformation of kinesin. These observations imply that when cargo is transported by both dynein and phosphorylated kinesin, a common occurrence in the cell, there may be a bias that favors motion toward the minus-end of microtubules. Such bias could be used to tune transport in healthy cells when properly regulated but contribute to a disease state when misregulated.     

An Unbiased Approach to Identifying Tau Kinases That Phosphorylate Tau at Sites Associated with Alzheimer Disease

Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.     

Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.     

The ancient tropical rainforest tree Symphonia globulifera L. f. (Clusiaceae) was not restricted to postulated Pleistocene refugia in Atlantic Equatorial Africa

Understanding the history of forests and their species'' demographic responses to past disturbances is important for predicting impacts of future environmental changes. Tropical rainforests of the Guineo-Congolian region in Central Africa are believed to have survived the Pleistocene glacial periods in a few major refugia, essentially centred on mountainous regions close to the Atlantic Ocean. We tested this hypothesis by investigating the phylogeographic structure of a widespread, ancient rainforest tree species, Symphonia globulifera L. f. (Clusiaceae), using plastid DNA sequences (chloroplast DNA [cpDNA], psbA-trnH intergenic spacer) and nuclear microsatellites (simple sequence repeats, SSRs). SSRs identified four gene pools located in Benin, West Cameroon, South Cameroon and Gabon, and São Tomé. This structure was also apparent at cpDNA. Approximate Bayesian Computation detected recent bottlenecks approximately dated to the last glacial maximum in Benin, West Cameroon and São Tomé, and an older bottleneck in South Cameroon and Gabon, suggesting a genetic effect of Pleistocene cycles of forest contraction. CpDNA haplotype distribution indicated wide-ranging long-term persistence of S. globulifera both inside and outside of postulated forest refugia. Pollen flow was four times greater than that of seed in South Cameroon and Gabon, which probably enabled rapid population recovery after bottlenecks. Furthermore, our study suggested ecotypic differentiation—coastal or swamp vs terra firme—in S. globulifera. Comparison with other tree phylogeographic studies in Central Africa highlighted the relevance of species-specific responses to environmental change in forest trees.