Proteostasis in neurodegenerative diseases.

Proteostasis is essential for normal function of proteins and vital for cellular health and survival. Proteostasis encompasses all stages in the "life" of a protein, that is, from translation to functional performance and, ultimately, to degradation. Proteins need native conformations for function and in the presence of multiple types of stress, their misfolding and aggregation can occur. A coordinated network of proteins is at the core of proteostasis in cells. Among these, chaperones are required for maintaining the integrity of protein conformations by preventing misfolding and aggregation and guide those with abnormal conformation to degradation. The ubiquitin-proteasome system (UPS) and autophagy are major cellular pathways for degrading proteins. Although failure or decreased functioning of components of this network can lead to proteotoxicity and disease, like neuron degenerative diseases, underlying factors are not completely understood. Accumulating misfolded and aggregated proteins are considered major pathomechanisms of neurodegeneration. In this chapter, we have described the components of three major branches required for proteostasis-chaperones, UPS and autophagy, the mechanistic basis of their function, and their potential for protection against various neurodegenerative conditions, like Alzheimer's, Parkinson's, and Huntington's disease. The modulation of various proteostasis network proteins, like chaperones, E3 ubiquitin ligases, proteasome, and autophagy-associated proteins as therapeutic targets by small molecules as well as new and unconventional approaches, shows promise.

Bayesian analysis dissects kinetic modulation during non-stationary gene expression.

Labelling of nascent stem loops with fluorescent proteins has fostered the visualization of transcription in living cells. Quantitative analysis of recorded fluorescence traces can shed light on kinetic transcription parameters and regulatory mechanisms. However, existing methods typically focus on steady state dynamics. Here, we combine a stochastic process transcription model with a hierarchical Bayesian method to infer global as well locally shared parameters for groups of cells and recover unobserved quantities such as initiation times and polymerase loading of the gene. We apply our approach to the cyclic response of the yeast CUP1 locus to heavy metal stress. Within the previously described slow cycle of transcriptional activity on the scale of minutes, we discover fast time-modulated bursting on the scale of seconds. Model comparison suggests that slow oscillations of transcriptional output are regulated by the amplitude of the bursts. Several polymerases may initiate during a burst.

Electrode impedances in children with cochlear implants: Comparison between intra-operative Switch ON and post-operative Switch ON.

INTRODUCTION: Intra-operative Switch ON (IOSO) is a novel clinical approach of activating the cochlear implant during the surgery adopted at our cochlear implantation center.We compared the electrode impedances in two conditions of Switch ON of cochlear implants; IOSO and post-operative Switch ON (POSO, 21st day of surgery). METHODS: Electrode impedances of 185 cochlear implants, 93 of whom received IOSO and 92 POSO, recorded over 10 years were analyzed retrospectively. RESULTS: Electrode impedances of IOSO group were significantly lower than POSO group at Switch ON and 3rd, 6th, 9th, and 12th months post cochlear implantation. In IOSO group, 3rd month's electrode impedances were high when compared to electrode impedances at Switch ON. Beyond the 3rd months, electrode impedance remained unchanged. In POSO group, there were no significant differences in electrode impedances between any measurement schedule. CONCLUSIONS: To our knowledge, this is the first study to investigate in detail the electrode impedances of the two above-said conditions of Switch ON in the process of cochlear implantation. This study concludes that timing of CI Switch ON has a significant effect on the electrode impedances. These results may affect the choice of cochlear implant Switch ON timing.

Single-molecule tracking dataset of histone H3 (Hht1) in Saccharomyces cerevisiae.

Single-Molecule Tracking (SMT) is a powerful method to quantify protein dynamics in live cells. Recently, we have established a data analysis pipeline for estimating various biophysical parameters (mean squared displacement, diffusion coefficient, bound fraction, residence time, jump distances, jump angles, and track statistics) from the single-molecule time-lapse movies acquired from yeast Saccharomyces cerevisiae. We acquired the time-lapse movies using different time intervals (i.e. 15 ms, 200 ms, and 1000 ms) to extract the diffusion parameters (from 15 ms time interval movies) and residence time (from 200 ms and 1000 ms time interval movies). We tracked the single molecules from these movies using three MATLAB-based software packages (MatlabTrack, TrackIT, DiaTrack (Sojourner, and Spot-On)) to quantify various biophysical parameters. In this article, we have quantified the biophysical parameters of chromatin-bound histone H3 (Hht1), labeled using JF646 HaloTag Ligand (HTL), and shared a few raw time-lapse SMT movies for the same. Histone H3 is a chromatin-bound protein and it serves as a benchmark for the stably bound molecules for the SMT experiments. Hence, this dataset can be used by various researchers to quantify the biophysical parameters of chromatin-bound molecules (Histone H3). Any newly developed tracking software can use this dataset to validate the accuracy of its tracking algorithms.

Minichromosome maintenance proteins in eukaryotic chromosome segregation.

Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.

Single-molecule tracking for studying protein dynamics and target-search mechanism in live cells of S. cerevisiae.

Single-molecule tracking (SMT) is a powerful approach to quantify the biophysical parameters of protein dynamics in live cells. Here, we describe a protocol for SMT in live cells of the budding yeast Saccharomyces cerevisiae. We detail how to genetically engineer yeast strains for SMT, how to set up image acquisition parameters, and how different software programs can be used to quantify a variety of biophysical parameters such as diffusion coefficient, residence time, bound fraction, jump angles, and target-search parameters. For complete details on the use and execution of this protocol, please refer to Mehta et al. 1 and Ball et al..2.

In-vivo Single-Molecule Imaging in Yeast: Applications and Challenges.

Single-molecule imaging has gained momentum to quantify the dynamics of biomolecules in live cells, as it provides direct real-time measurements of various cellular activities under their physiological environment. Yeast, a simple and widely used eukaryote, serves as a good model system to quantify single-molecule dynamics of various cellular processes because of its low genomic and cellular complexities, as well as its facile ability to be genetically manipulated. In the past decade, significant developments have been made regarding the intracellular labeling of biomolecules (proteins, mRNA, fatty acids), the microscopy setups to visualize single-molecules and capture their fast dynamics, and the data analysis pipelines to interpret such dynamics. In this review, we summarize the current state of knowledge for the single-molecule imaging in live yeast cells to provide a ready reference for beginners. We provide a comprehensive table to demonstrate how various labs tailored the imaging regimes and data analysis pipelines to estimate various biophysical parameters for a variety of biological processes. Lastly, we present current challenges and future directions for developing better tools and resources for single-molecule imaging in live yeast cells.

Streamlined Ruthenium(II) Catalysis for One-Pot 2-fold Unsymmetrical C-H Olefination of (Hetero)Arenes.

The potential of carboxylate-directed Ru(II)-catalyzed C-H bond activation has been exploited in the first example of challenging 2-fold unsymmetrical cross-dehydrogenative olefination of (hetero)aromatic carboxylic acids. This highly regioselective protocol operates in one pot under a single catalytic system and tolerates a wide spectrum of carboxylic acids as well as activated olefins. A rare example of an unsymmetrical meta-bis-olefination approach has also been successfully engineered via a tailored unsymmetrical double C-H functionalization and concomitant decarboxylation process.

Single-Molecule Analysis Reveals Linked Cycles of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast.

It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.

An interplay between Shugoshin and Spo13 for centromeric cohesin protection and sister kinetochore mono-orientation during meiosis I in Saccharomyces cerevisiae.

Meiosis is a specialized cell division process by which haploid gametes are produced from a diploid mother cell. Reductional chromosome segregation during meiosis I (MI) is achieved by two unique and conserved events: centromeric cohesin protection (CCP) and sister kinetochore mono-orientation (SKM). In Saccharomyces cerevisiae, a meiosis-specific protein Spo13 plays a role in both these centromere-specific events. Despite genome-wide association of Spo13, we failed to detect its function in global processes such as cohesin loading, cohesion establishment and homologs pairing. While Shugoshin (Sgo1) and protein phosphatase 2A (PP2ARts1) play a central role in CCP, it is not fully understood whether Spo13 functions in the process through a Sgo1- PP2ARts1-dependent or -independent mechanism. To delineate this and to find the relative contribution of each of these proteins in CCP and SKM, we meticulously observed the sister chromatid segregation pattern in the wild type, sgo1Δ, rts1Δ and spo13Δ single mutants and in their respective double mutants. We found that Spo13 protects centromeric cohesin through a Sgo1- PP2ARts1-independent mechanism. To our surprise, we observed a hitherto unknown role of Sgo1 in SKM. Further investigation revealed that Sgo1-mediated recruitment of aurora kinase Ipl1 to the centromere facilitates monopolin loading at the kinetochore during MI. Hence, this study uncovers the role of Sgo1 in SKM and demonstartes how the regulators (Sgo1, PP2ARts1, Spo13) work in a coordinated manner to achieve faithful chromosome segregation during meiosis, the failure of which leads to aneuploidy and birth defects.

Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin.

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

Role of Ctf3 and COMA subcomplexes in meiosis: Implication in maintaining Cse4 at the centromere and numeric spindle poles.

During mitosis and meiosis, kinetochore, a conserved multi-protein complex, connects microtubule with the centromere and promotes segregation of the chromosomes. In budding yeast, central kinetochore complex named Ctf19 has been implicated in various functions and is believed to be made up of three biochemically distinct subcomplexes: COMA, Ctf3 and Iml3-Chl4. In this study, we aimed to identify whether Ctf3 and COMA subcomplexes have any unshared function at the kinetochore. Our data suggests that both these subcomplexes may work as a single functional unit without any unique functions, which we tested. Analysis of severity of the defects in the mutants suggests that COMA is epistatic to Ctf3 subcomplex. Interestingly, we noticed that these subcomplexes affect the organization of mitotic and meiotic kinetochores with subtle differences and they promote maintenance of Cse4 at the centromeres specifically during meiosis which is similar to the role of Mis6 (Ctf3 homolog) in fission yeast during mitosis. Interestingly, analysis of ctf3Δ and ctf19Δ mutants revealed a novel role of Ctf19 complex in regulation of SPB cohesion and duplication in meiosis.

Functional characterization of kinetochore protein, Ctf19 in meiosis I: an implication of differential impact of Ctf19 on the assembly of mitotic and meiotic kinetochores in Saccharomyces cerevisiae.

Meiosis is a specialized cell division process through which chromosome numbers are reduced by half for the generation of gametes. Kinetochore, a multiprotein complex that connects centromeres to microtubules, plays essential role in chromosome segregation. Ctf19 is the key central kinetochore protein that recruits all the other non-essential proteins of the Ctf19 complex in budding yeast. Earlier studies have shown the role of Ctf19 complex in enrichment of cohesin around the centromeres both during mitosis and meiosis, leading to sister chromatid cohesion and meiosis II disjunction. Here we show that Ctf19 is also essential for the proper execution of the meiosis I specific unique events, such as non-homologous centromere coupling, homologue pairing, chiasmata resolution and proper orientation of homologues and sister chromatids with respect to the spindle poles. Additionally, this investigation reveals that proper kinetochore function is required for faithful chromosome condensation in meiosis. Finally, this study suggests that absence of Ctf19 affects the integrity of meiotic kinetochore differently than that of the mitotic kinetochore. Consequently, absence of Ctf19 leads to gross chromosome missegregation during meiosis as compared with mitosis. Hence, this study reports for the first time the differential impact of a non-essential kinetochore protein on the mitotic and meiotic kinetochore ensembles and hence chromosome segregation.

Cohesin: functions beyond sister chromatid cohesion.

Faithful segregation of chromosomes during mitosis and meiosis is the cornerstone process of life. Cohesin, a multi-protein complex conserved from yeast to human, plays a crucial role in this process by keeping the sister chromatids together from S-phase to anaphase onset during mitosis and meiosis. Technological advancements have discovered myriad functions of cohesin beyond its role in sister chromatid cohesion (SCC), such as transcription regulation, DNA repair, chromosome condensation, homolog pairing, monoorientation of sister kinetochore, etc. Here, we have focused on such functions of cohesin that are either independent of or dependent on its canonical role of sister chromatid cohesion. At the end, human diseases associated with malfunctioning of cohesin, albeit with mostly unperturbed sister chromatid cohesion, have been discussed.

Iml3p, a component of the Ctf19 complex of the budding yeast kinetochore is required to maintain kinetochore integrity under conditions of spindle stress.

The Ctf19 multi-protein complex of the central kinetochore in Saccharomyces cerevisiae is required for precise chromosome segregation during mitosis. Of 11 proteins of this complex, at least six are required for cell survival when microtubules are defective. To find individual roles of these proteins in kinetochore stability, double deletion mutants of the corresponding genes were constructed in several combinations. The growth phenotype of all the mutants was in accordance with the current model of hierarchical assembly of kinetochore proteins except one that lacked CHL4 and IML3 genes in a tubulin-defective background (tub1-1 chl4 iml3). tub1-1 chl4 iml3 showed synergistic growth defect, decrease in minichromosome stability compared with its single mutants, and a greater accumulation of cells at the G2/M checkpoint of the cell cycle. Furthermore, in the absence of Iml3p, the two-hybrid interaction between Ctf19p (a member of the Ctf19 complex) and Dam1p (a member of the outer kinetochore DASH complex) was disrupted and the localization of Dam1p at the kinetochore was also compromised. These results indicate a role for Iml3p distinct from Chl4p at the kinetochore. Iml3p may be acting as a link between the central and the outer complexes thus contributing to a functional kinetochore.

Cohesin: a guardian of genome integrity.

Ability to reproduce is one of the hallmark features of all life forms by which new organisms are produced from their progenitors. During this process each cell duplicates its genome and passes a copy of its genome to the daughter cells along with the cellular matrix. Unlike bacteria, in eukaryotes there is a definite time gap between when the genome is duplicated and when it is physically separated. Therefore, for precise halving of the duplicated genome into two, it is required that each pair of duplicated chromosomes, termed sister chromatids, should be paired together in a binary fashion from the moment they are generated. This pairing function between the duplicated genome is primarily provided by a multimeric protein complex, called cohesin. Thus, genome integrity largely depends on cohesin as it ensures faithful chromosome segregation by holding the sister chromatids glued together from S phase to anaphase. In this review, we have discussed the life cycle of cohesin during both mitotic and meiotic cell divisions including the structure and architecture of cohesin complex, relevance of cohesin associated proteins, mechanism of cohesin loading onto the chromatin, cohesion establishment and the mechanism of cohesin disassembly during anaphase to separate the sister chromatids. We have also focused on the role of posttranslational modifications in cohesin biology. For better understanding of the complexity of the cohesin regulatory network to the readers, we have presented an interactome profiling of cohesin core subunits in budding yeast during mitosis and meiosis.

Centromere identity: a challenge to be faced.

The centromere is a genetic locus, required for faithful chromosome segregation, where spindle fibers attach to the chromosome through kinetochore. Loss of centromere or formation of multiple centromeres on a single chromosome leads to chromosome missegregation or chromosome breakage, respectively, which are detrimental for fitness and survival of a cell. Therefore, understanding the mechanism of centromere locus determination on the chromosome and perpetuation of such a locus in subsequent generation (known as centromere identity) is very fundamental to combat conditions like aneuploidy, spontaneous abortion, developmental defects, cell lethality and cancer. Recent studies have come up with different models to explain centromere identity. However, the exact mechanism still remains elusive. It has been observed that most eukaryotic centromeres are determined epigenetically rather than by a DNA sequence. The epigenetic marks that are instrumental in determining centromere identity are the histone H3 variant, CENP-A and the specialized posttranslational modification of the core histones. Here we will review the recent studies on the factors responsible for generating unique centromeric chromatin and how it perpetuates during cell division giving the present-day models. We will further focus on the probable mechanism of de novo centromere formation with an example of neocentromere. As a matter of similitude, this review will include marking extrachromosomal chromatin to be served as a partitioning locus by deposition of CENP-A homolog in budding yeast.

Label-free detection techniques for protein microarrays: prospects, merits and challenges.

Protein microarrays, on which thousands of discrete proteins are printed, provide a valuable platform for functional analysis of the proteome. They have been widely used for biomarker discovery and to study protein-protein interactions. The accomplishments of DNA microarray technology, which had enabled massive parallel studies of gene expression, sparked great interest for the development of protein microarrays to achieve similar success at the protein level. Protein microarray detection techniques are often classified as being label-based and label-free. Most of the microarray applications have employed labelled detection such as fluorescent, chemiluminescent and radioactive labelling. These labelling strategies have synthetic challenges, multiple label issues and may exhibit interference with the binding site. Therefore, development of sensitive, reliable, high-throughput, label-free detection techniques are now attracting significant attention. Label-free detection techniques monitor biomolecular interactions and simplify the bioassays by eliminating the need for secondary reactants. Moreover, they provide quantitative information for the binding kinetics. In this article, we will review several label-free techniques, which offer promising applications for the protein microarrays, and discuss their prospects, merits and challenges.