Search

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.

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.

Summary 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.

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.

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.

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.

Aims: Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Methods and Results: Urea, K2HPO4, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l−1): urea, 0·33; K2HPO4, 1·17; MgSO4, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml−1 of chitinase. Conclusions: The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K2HPO4, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production. Significance and Impact of the Study: The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.

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.

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.

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.

The Extracellular Matrix (ECM) undergoes changes in composition and organization during tumor progression. In breast cancer, increased deposition and cross linking-induced alignment of collagen I create a stiffer microenvironment that directly contributes to cancer invasion. While ECM stiffness-induced invasion has been documented, it remains unclear if ECM density also contributes to invasion independent of ECM stiffness. In this paper, using collagen I-coated glass coverslips of varying density, we sought to study the influence of ECM density on the invasiveness of human MDA-MB-231 breast cancer cells. We first showed that cell spreading and contractility increases with ECM density. Concomitant with increase in cell contractility, matrix degradation was seen to increase with ECM density and was associated with higher invadopodia activity. The density-dependent increase in degradation was associated with higher activity of MMP-2, MMP-9 and MT1-MMP. Treatment with either the MMP inhibitor GM6001 or the myosin II inhibitor blebbistatin, were found to inhibit cell contractility and suppress matrix degradation. Contractility was found to modulate the activity of MMP-2 and MMP-9, and the localization of MT1-MMP at invadopodia. Taken together, our results indicate that ECM density regulates ECM degradation through modulation of cell contractility.

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.