Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Estimating the Cholesterol Affinity of Integral Membrane Proteins from Experimental Data.

The cholesterol affinities of many integral plasma membrane proteins have been estimated by molecular computation. However, these values lack experimental confirmation. We therefore developed a simple mathematical model to extract sterol affinity constants and stoichiometries from published isotherms for the dependence of the activity of such proteins on the membrane cholesterol concentration. The binding curves for these proteins are sigmoidal, with strongly lagged thresholds attributable to competition for the cholesterol by bilayer phospholipids. The model provided isotherms that matched the experimental data using published values for the sterol association constants and stoichiometries of the phospholipids. Three oligomeric transporters were found to bind cholesterol without cooperativity, with dimensionless association constants of 35 for Kir3.4* and 100 for both Kir2 and a GAT transporter. (The corresponding ΔG° values were -8.8, -11.4, and -11.4 kJ/mol, respectively). These association constants are significantly lower than those for the phospholipids, which range from ∼100 to 6000. The BK channel, the nicotinic acetylcholine receptor, and the M192I mutant of Kir3.4* appear to bind multiple cholesterol molecules cooperatively (n = 2 or 4), with subunit affinities of 563, 950, and 700, respectively. The model predicts that the three less avid transporters are approximately half-saturated in their native plasma membranes; hence, they are sensitive to variations in cholesterol in vivo. The more avid proteins would be nearly saturated in vivo. The method can be applied to any integral protein or other ligands in any bilayer for which there are reasonable estimates of the sterol affinities and stoichiometries of the phospholipids.

Protooncogenic Role of ARHGAP11A and ARHGAP11B in Invasive Ductal Carcinoma: Two Promising Breast Cancer Biomarkers.

Invasive duct carcinoma (IDC) is one of the most common types of breast cancer (BC) in women worldwide, with a high risk of malignancy, metastasis, recurrence, and death. So far, molecular patterns among IDC cases have not been fully defined. However, extensive evidence has shown that dysregulated Rho family small GTPases (Rho GTPases) including Rho GTPase activating proteins (RhoGAPs) have important roles in the invasive features of IDCs. In the current study, we analyzed the expression levels of two RhoGAP genes, ARHGAP11A and ARHGAP11B, in The Cancer Genome Atlas (TCGA) breast cancer (BRCA) and also our 51 IDC tumors compared to their matched normal tissues using quantitative polymerase chain reaction (qPCR). Our TCGA data analysis revealed higher expression of ARHGAP11A and ARHGAP11B in various cancers comprising BCs. Also, we found correlations between these genes and other genes in TCGA-BRCA. Moreover, our methylation analysis showed that their promotor methylation had a negative correlation with their overexpression. QPCR revealed their significant upregulation in our tumor samples. Furthermore, we found that the expression level of ARHGAP11A was considerably lower in women who were breastfeeding. Moreover, it had overexpression in cases who had regular menstrual cycles and early age (younger than 14) at menarche. However, ARHGAP11B had a higher expression in HER2-positive tumors versus HER2-positive and ER-positive tumors. Our study found possible protooncogenic roles for these genes and their involvement in IDC pathogenesis and malignancy. Therefore, they can be considered novel prognostic and diagnostic biomarkers for IDC.

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding.

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.

Clinical values of two estrogen receptor signaling targeted lncRNAs in invasive ductal breast carcinoma.

BACKGROUND: Invasive ductal carcinoma (IDC) is the most frequent type of breast cancer (BC) in women, with a high clinical burden due to its high invasive properties. Despite of quickly emerging new data regarding the molecular heterogeneity of invasive cancers, far less is known about the molecular patterns among cases of IDC. An expanding body of evidence has demonstrated that dysregulation of long noncoding RNAs (lncRNAs) is involved in the heterogeneity feature of BC. METHODS: In this study, we analyzed the expression levels of two novel lncRNAs LOC100288637 and RP11-48B3 in 51 IDC tissues in comparison with adjacent non-cancerous tissues. And finally, bio-informatic evaluation has been done. RESULTS: The results of quantitative polymerase chain reaction showed that LOC100288637 and RP11-48B3 were significantly overexpressed in tumor tissues compared to normal samples (P = 0.0085 and P = 0.0002, respectively). Also, the two lncRNAs were overexpressed in both MDA-MB-231 and MCF-7 BC cell lines, nevertheless, with a higher expression pattern in MDA-MB-231 than MCF7 cell line. Furthermore, LOC100288637 had an elevated expression level in HER-2 positive tumors compared to HER-2 negative tumors (P = 0.031). Interestingly, the lncRNA RP11-48B3.4 was upregulated in IDC subjects with the age at menarche < 14 years compared to patients with the age at menarche 14 (P = 0.041). It was observed in another result that lncRNA RP11-48B3.4 is significantly upregulated in tumors with a lower histological grade compared to tumor samples with higher grades (P = 0.047). And finally, using bio-informatic evaluation, we found a predicted interaction between RP11-48B3.4 and mRNA zinc finger and BTB domain containing 10 (ZBTB10). CONCLUSION: Altogether, our findings suggest that these lncRNAs with potential oncogenic roles are involved in the pathogenesis of IDC with clinical significance and they may therefore serve as novel markers for the dia-gnosis and treatment of IDC.

A basic model for cell cholesterol homeostasis.

Cells manage their cholesterol by negative feedback using a battery of sterol-responsive proteins. How these activities are coordinated so as to specify the abundance and distribution of the sterol is unclear. We present a simple mathematical model that addresses this question. It assumes that almost all of the cholesterol is associated with phospholipids in stoichiometric complexes. A small fraction of the sterol is uncomplexed and thermodynamically active. It equilibrates among the organelles, setting their sterol level according to the affinity of their phospholipids. The activity of the homeostatic proteins in the cytoplasmic membranes is then set by their fractional saturation with uncomplexed cholesterol in competition with the phospholipids. The high-affinity phospholipids in the plasma membrane (PM) are filled to near stoichiometric equivalence, giving it most of the cell sterol. Notably, the affinity of the phospholipids in the endomembranes (EMs) is lower by orders of magnitude than that of the phospholipids in the PM. Thus, the small amount of sterol in the EMs rests far below stoichiometric capacity. Simulations match a variety of experimental data. The model captures the essence of cell cholesterol homeostasis, makes coherent a diverse set of experimental findings, provides a surprising prediction and suggests new experiments.

KERA: analysis tool for multi-process, multi-state single-molecule data.

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA's utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.

Distinguishing synaptic vesicle precursor navigation of microtubule ends with a single rate constant model.

Axonal motor driven cargo utilizes the microtubule cytoskeleton in order to direct cargo, such as synaptic vesicle precursors (SVP), to where they are needed. This transport requires vesicles to travel up to microns in distance. It has recently been observed that finite microtubule lengths can act as roadblocks inhibiting SVP and increasing the time required for transport. SVPs reach the end of a microtubule and pause until they can navigate to a neighboring microtubule in order to continue transport. The mechanism(s) by which axonal SVPs navigate the end of a microtubule in order to continue mobility is unknown. In this manuscript we model experimentally observed vesicle pausing at microtubule ends in C. elegans. We show that a single rate-constant model reproduces the time SVPs pause at MT-ends. This model is based on the time an SVP must detach from its current microtubule and re-attach to a neighboring microtubule. We show that vesicle pause times are different for anterograde and retrograde motion, suggesting that vesicles utilize different proteins at plus and minus end sites. Last, we show that vesicles do not likely utilize a tug-of-war like mechanism and reverse direction in order to navigate microtubule ends.

Effects of Intercalating Molecules on the Polymer Properties of DNA.

Atomic force microscopy (AFM) enables determination of physical properties from single DNA molecules. Insertion of aromatic molecules into the structure of DNA results in morphological changes. However, the accompanying changes to elastic properties due to this insertion are not fully understood. AFM was used to examine the morphological effects of intercalator binding and report changes in the elastic properties of intrinsically straight DNA molecules. The persistence length and polymer extension were characterized in the presence of three intercalating molecules: ethidium bromide and the less well studied chloroquine and acridine. It was found that all three intercalators significantly increased the bending persistence length. In addition, an analysis of the normal bending modes of the static molecules corroborated these results. This approach of measuring binding effects of intercalators on DNA physical properties using a model system of intrinsically straight DNA is applicable to other DNA binding ligands and other modes of DNA interaction.

Dynamic motions of molecular motors in the actin cytoskeleton.

During intracellular transport, cellular cargos, such as organelles, vesicles, and proteins, are transported within cells. Intracellular transport plays an important role in diverse cellular functions. Molecular motors walking on the cytoskeleton facilitate active intracellular transport, which is more efficient than diffusion-based passive transport. Active transport driven by kinesin and dynein walking on microtubules has been studied well during recent decades. However, mechanisms of active transport occurring in disorganized actin networks via myosin motors remain elusive. To provide physiologically relevant insights, we probed motions of myosin motors in actin networks under various conditions using our well-established computational model that rigorously accounts for the mechanical and dynamical behaviors of the actin cytoskeleton. We demonstrated that myosin motions can be confined due to three different reasons in the absence of F-actin turnover. We verified mechanisms of motor stalling using in vitro reconstituted actomyosin networks. We also found that with F-actin turnover, motors consistently move for a long time without significant confinement. Our study sheds light on the importance of F-actin turnover for effective active transport in the actin cytoskeleton.

Anomalous intracellular transport phases depend on cytoskeletal network features.

Intracellular transport in eukaryotic cells consists of phases of passive, diffusion-based transport and active, motor-driven transport along filaments that make up the cell's cytoskeleton. The interplay between superdiffusive transport along cytoskeletal filaments and the anomalous nature of subdiffusion in the bulk can lead to novel effects in transport behavior at the cellular scale. Here we develop a computational model of the process with cargo being ballistically transported along explicitly modeled cytoskeletal filament networks and passively transported in the cytoplasm by a subdiffusive continuous-time random walk (CTRW). We show that, over a physiologically relevant range of filament lengths and numbers, the network introduces a filament-length sensitive superdiffusive phase at early times which crosses over to a phase where the CTRW is dominant and produces subdiffusion at late times. We apply our approach to the problem of insulin secretion from cells and show that the superdiffusive phase introduced by the filament network manifests as a peak in the secretion at early times followed by an extended sustained release phase that is dominated by the CTRW process at late times. Our results are consistent with in vivo observations of insulin transport in healthy cells and shed light on the potential for the cell to tune functionally important transport phases by altering its cytoskeletal network.

Dynamics and selective remodeling of the DNA-binding domains of RPA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

Entropy production rate is maximized in non-contractile actomyosin.

The actin cytoskeleton is an active semi-flexible polymer network whose non-equilibrium properties coordinate both stable and contractile behaviors to maintain or change cell shape. While myosin motors drive the actin cytoskeleton out-of-equilibrium, the role of myosin-driven active stresses in the accumulation and dissipation of mechanical energy is unclear. To investigate this, we synthesize an actomyosin material in vitro whose active stress content can tune the network from stable to contractile. Each increment in activity determines a characteristic spectrum of actin filament fluctuations which is used to calculate the total mechanical work and the production of entropy in the material. We find that the balance of work and entropy does not increase monotonically and the entropy production rate is maximized in the non-contractile, stable state of actomyosin. Our study provides evidence that the origins of entropy production and activity-dependent dissipation relate to disorder in the molecular interactions between actin and myosin.

Improved statistical methods enable greater sensitivity in rhythm detection for genome-wide data.

Robust methods for identifying patterns of expression in genome-wide data are important for generating hypotheses regarding gene function. To this end, several analytic methods have been developed for detecting periodic patterns. We improve one such method, JTK_CYCLE, by explicitly calculating the null distribution such that it accounts for multiple hypothesis testing and by including non-sinusoidal reference waveforms. We term this method empirical JTK_CYCLE with asymmetry search, and we compare its performance to JTK_CYCLE with Bonferroni and Benjamini-Hochberg multiple hypothesis testing correction, as well as to five other methods: cyclohedron test, address reduction, stable persistence, ANOVA, and F24. We find that ANOVA, F24, and JTK_CYCLE consistently outperform the other three methods when data are limited and noisy; empirical JTK_CYCLE with asymmetry search gives the greatest sensitivity while controlling for the false discovery rate. Our analysis also provides insight into experimental design and we find that, for a fixed number of samples, better sensitivity and specificity are achieved with higher numbers of replicates than with higher sampling density. Application of the methods to detecting circadian rhythms in a metadataset of microarrays that quantify time-dependent gene expression in whole heads of Drosophila melanogaster reveals annotations that are enriched among genes with highly asymmetric waveforms. These include a wide range of oxidation reduction and metabolic genes, as well as genes with transcripts that have multiple splice forms.

Effect of vitamins A, E, C and omega-3 fatty acids supplementation on the level of catalase and superoxide dismutase activities in streptozotocin-induced diabetic rats.

BACKGROUND: Since free radicals and antioxidant enzymes may play an important role in the development of diabetes, the present study was designed to assess the effect of supplementation with vitamins A, E and C and ω-3 fatty acids on catalase and superoxide dismutase activity in streptozotocin (STZ)-induced diabetic rats. METHODS: A total of 64 male Wistar rats weighing 250 g were divided into four groups as normal control, diabetic control, diabetic supplemented with vitamin A, E and C and diabetic supplemented with ω-3 fatty acids. After four weeks the rats were anesthetized and catalase (CAT) and superoxide dismutase (SOD) activities were investigated in blood samples, liver and heart homogenates. RESULTS: In diabetic rats, the activity levels of heart SOD (p < 0.001) and heart and liver CAT (p < 0.001) were significantly lower than in normal control rats. Supplementation with vitamins A, E and C significantly increased heart CAT (p = 0.05). No significant change was observed in diabetic rats supplemented with ω-3 fatty acids. CONCLUSION: Supplementation with vitamins A, E and C and ω-3 fatty acids was found to increase heart CAT activity in diabetic rats and they can be valuable candidates in the treatment of the complications of diabetes (Tab. 6, Ref. 26).

Simple renal cysts and hypertension are associated with angiotensinogen (AGT) gene variant in Shiraz population (Iran).

AIM: To our knowledge, the relationship between simple renal cysts, hypertension and three significant genes of the renin-angiotensin system (AGT, AT1R and ACE1) has not been studied. The present study was designed to search for possible relationships between these significant polymorphic components, hypertension and simple renal cysts in Shiraz province (Iran). METHODS: A total of 160 participants were recruited from the Motahari Clinic at Shiraz University of Medical Sciences. The subjects were divided into four main groups. Detection of the ACE1 genotype was performed with a nested-polymerase chain reaction (PCR) protocol. Two separate restriction fragment length polymorphism-PCR assays were used to identify AGT and AT1R genotypes. RESULTS: The allele frequency of AGT M235T differed significantly between group 1 (patients with simple renal cysts and hypertension) and normal individuals (p < 0.05). There were no significant differences in frequency for the other genes (ACE1 and AT1R). CONCLUSIONS: Our findings show a relationship between the AGT-TT genotype and hypertension in patients with both hypertension and simple renal cysts. This finding suggests an additive role for the AGT gene of the renin-angiotensin system in the process of hypertension and simple renal cysts formation. Future studies are needed to elucidate the mechanisms through which this association is mediated.

Microtubule orientation and spacing within bundles is critical for long-range kinesin-1 motility.

Cells rely on active transport to quickly organize cellular cargo. How cells regulate transport is not fully understood. One proposed mechanism is that motor activity could be altered through the architecture of the cytoskeleton. This mechanism is supported by the fact that the cytoskeletal network is tightly regulated in cells and filament polarity within networks dictates motor directionality. For instance, axons contain bundles of parallel microtubules and all cargos with the same motor species will move in the same direction. It is not clear how other types of networks, such as antiparallel bundles in dendrites, can regulate motor transport. To understand how the organization of microtubules within bundles can regulate transport, we studied kinesin-1 motility on three bundle types: random-polarity bundles that are close-packed, parallel polarity bundles, and antiparallel polarity bundles that are spaced apart. We find that close-packed bundles inhibit motor motion, while parallel arrays support unidirectional motion. Spacing the microtubules with microtubule-associated proteins enhances run lengths. Our results indicate that microtubule bundle architecture dictates the motion of single motors and could have effects on cargo transport. © 2014 Wiley Periodicals, Inc.

Distribution of directional change as a signature of complex dynamics.

Analyses of random walks traditionally use the mean square displacement (MSD) as an order parameter characterizing dynamics. We show that the distribution of relative angles of motion between successive time intervals of random walks in two or more dimensions provides information about stochastic processes beyond the MSD. We illustrate the behavior of this measure for common models and apply it to experimental particle tracking data. For a colloidal system, the distribution of relative angles reports sensitively on caging as the density varies. For transport mediated by molecular motors on filament networks in vitro and in vivo, we discover self-similar properties that cannot be described by existing models and discuss possible scenarios that can lead to the elucidated statistical features.

Stability and stoichiometry of bilayer phospholipid-cholesterol complexes: relationship to cellular sterol distribution and homeostasis.

Is cholesterol distributed among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their capacity for association with the sterol. We examined this issue by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing phospholipids and varied levels of cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly 4 orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested cholesterol:phospholipid association ratios of 1:1, 2:3, or 1:2 (moles:moles). The accessibility of cholesterol above these thresholds was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the stoichiometries of the putative cholesterol-phospholipid complexes, their relative stabilities, and the fractions of bilayer cholesterol not in complexes at the threshold equivalence points. Predictably, the saturated phosphorylcholine species had the lowest apparent stoichiometric ratios and the strongest associations with cholesterol. These results are in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-ß-cyclodextrin. In addition, the behavior of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.