Cytoskeleton dynamics: fluctuations within the network

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.     

Directional memory and caged dynamics in cytoskeletal remodelling

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.     

Physicochemical sequence characteristics that influence S-palmitoylation propensity

Over the past 30 years, several hundred eukaryotic proteins spanning from yeast to man have been shown to be S-palmitoylated. This post-translational modification involves the reversible addition of a 16-carbon saturated fatty acyl chain onto the cysteine residue of a protein where it regulates protein membrane association and distribution, conformation, and stability. However, the large-scale proteome-wide discovery of new palmitoylated proteins has been hindered by the difficulty of identifying a palmitoylation consensus sequence. Using a bioinformatics approach, we show that the enrichment of hydrophobic and basic residues, the cellular context of the protein, and the structural features of the residues surrounding the palmitoylated cysteine all influence the likelihood of palmitoylation. We developed a new palmitoylation predictor that incorporates these identified features, and this predictor achieves a Matthews Correlation Coefficient of .74 using 10-fold cross validation, and significantly outperforms existing predictors on unbiased testing sets. This demonstrates that palmitoylation sites can be predicted with accuracy by taking into account not only physiochemical properties of the modified cysteine and its surrounding residues, but also structural parameters and the subcellular localization of the modified cysteine. This will allow for improved predictions of palmitoylated residues in uncharacterized proteins. A web-based version of this predictor is currently under development.     

Subliminal instrumental conditioning demonstrated in the human brain

How the brain uses success and failure to optimize future decisions is a long-standing question in neuroscience. One computational solution involves updating the values of context-action associations in proportion to a reward prediction error. Previous evidence suggests that such computations are expressed in the striatum and, as they are cognitively impenetrable, represent an unconscious learning mechanism. Here, we formally test this by studying instrumental conditioning in a situation where we masked contextual cues, such that they were not consciously perceived. Behavioral data showed that subjects nonetheless developed a significant propensity to choose cues associated with monetary rewards relative to punishments. Functional neuroimaging revealed that during conditioning cue values and prediction errors, generated from a computational model, both correlated with activity in ventral striatum. We conclude that, even without conscious processing of contextual cues, our brain can learn their reward value and use them to provide a bias on decision making.     

Free and bound state structures of 6-O-methyl homoerythromycins and epitope mapping of their interactions with ribosomes

The solution and solid state conformations of several 6-O-methyl homoerythromycins 1–4 were studied using a combination of X-ray crystallography, NMR spectroscopy and molecular modelling calculations. In the solid state 1 was found to exist as the two independent molecules with similar structures termed 3-endo-folded-out. In solution a significant conformational flexibility was noticed especially in the C2 to C5 region. The compounds 1 and 2 unlike 14-membered macrolides adopted the 3-endo-folded-out conformation while 3 and 4 existed in the classical folded-out conformation. TrNOESY and STD experiments showed that 1 and 2 bound to the Escherichia coli ribosome while 3 and 4, lacking the cladinose sugar, did not exhibit binding activities, this being in accordance with biochemical data. The bound conformations were found to be very similar to the free ones, some small differences were observed and discussed. The STD experiments provided evidence on binding epitopes. The structural parts of 1 and 2 in close contact with ribosome were similar, however the degree of saturation transfer was higher for 2. The differences between tr-NOE data and STD enhancements in 1 and 2 arouse as a consequence of structural changes upon binding and a closer proximity of 2 to the ribosome surface. An understanding of the molecular mechanisms involved in the interaction of macrolides with ribosomes can help in developing strategies aiming at design of potential inhibitors.     

Length-weight relationship in adult huchen Hucho hucho (L., 1758) from Drina River, Serbia

Huchen Hucho hucho from the Drina River (Serbia) revealed recently the decrease in breakpoint values for both standard length (Sl) and weight (w) in relation to the values recorded in 1999 from 110 cm to 98.4 cm and from 16.5 kg to 10.5 kg, respectively. That might indicate to the change in certain population parameters that could have an influence to the growth in the adult period of life.     

SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1–2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men.