scholarly journals Nanoscale structural organization and stoichiometry of the budding yeast kinetochore

2021 ◽  
Author(s):  
Konstanty Cieslinski ◽  
Yu-Le Wu ◽  
Lisa Neechyporenko ◽  
Sarah Janice Hoerner ◽  
Duccio Conti ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Molecule ◽  
Budding Yeast ◽  
Active Role ◽  
Quantitative Model ◽  
Binding Partner ◽  
Evolutionarily Conserved ◽  
Spindle Microtubules ◽  
Single Molecule Localization Microscopy

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multi-protein complex that physically links the DNA to spindle microtubules, and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions, and how they ensure an error-free outcome of mitosis, is still limited, partly because we lack a comprehensive understanding of the kinetochore structure in the cell. In this study, we use single molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For all major kinetochore proteins, we measured abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging of kinetochores, our results present a somewhat different but unifying model of the inner kinetochore. We find that the centromere-specialized histone Cse4 is present in more than two copies per kinetochore along with its binding partner Mif2.

scholarly journals Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

2020 ◽  
Vol 219 (4) ◽  
Author(s):  
Gisela Cairo ◽  
Anne M. MacKenzie ◽  
Soni Lacefield
Keyword(s):  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Mitotic Chromosome ◽  
Budding Yeast ◽  
Meiotic Chromosome ◽  
Protein Phosphatase 1 ◽  
Aurora B ◽  
Chromosome Missegregation ◽  
Anaphase Onset ◽  
Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

scholarly journals In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yusuke Nasu ◽  
Alexander Benke ◽  
Satoko Arakawa ◽  
Go J. Yoshida ◽  
Genki Kawamura ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Molecule ◽  
In Situ Characterization ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals The chromosomal passenger complex establishes chromosome biorientation via two parallel localization pathways

2020 ◽  
Author(s):  
Theodor Marsoner ◽  
Poornima Yedavalli ◽  
Chiara Masnovo ◽  
Katrin Schmitzer ◽  
Christopher S. Campbell
Keyword(s):  
Chromosome Segregation ◽  
Budding Yeast ◽  
Small Region ◽  
The Other ◽  
Chromosomal Passenger Complex ◽  
Microtubule Binding ◽  
Chromosome Alignment ◽  
Chromosomal Passenger ◽  
Spindle Microtubules ◽  
Do So

AbstractChromosome biorientation is established by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore-microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore and spindle microtubules. Here we show that a small region of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This region, the SAH, is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of these three locations individually, we found that inner centromere localization is sufficient to establish chromosome biorientation and viability independently of the other two targeting mechanisms. Remarkably, although neither the inner kinetochore nor microtubule binding activities was able to rescue viability individually, they were able to do so when combined. We have therefore identified two parallel pathways by which the CPC can promote chromosome biorientation and proper completion of mitosis.

scholarly journals mRNA detection in budding yeast with single fluorophores

2017 ◽  
Author(s):  
Gable M. Wadsworth ◽  
Rasesh Y. Parikh ◽  
John S. Choy ◽  
Harold D. Kim
Keyword(s):  
Single Molecule ◽  
Budding Yeast ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Isoform

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect more than 50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

scholarly journals The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

2003 ◽  
Vol 160 (3) ◽  
pp. 329-339 ◽  
Author(s):  
Stéphanie Buvelot ◽  
Sean Y. Tatsutani ◽  
Danielle Vermaak ◽  
Sue Biggins
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Genomic Stability ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Spindle Disassembly ◽  
Spindle Midzone ◽  
Spindle Microtubules

Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

scholarly journals Structural plasticity of the living kinetochore

2017 ◽  
Vol 216 (11) ◽  
pp. 3551-3570 ◽  
Author(s):  
Karthik Dhatchinamoorthy ◽  
Manjunatha Shivaraju ◽  
Jeffrey J. Lange ◽  
Boris Rubinstein ◽  
Jay R. Unruh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Structure ◽  
Chromosome Segregation ◽  
Budding Yeast ◽  
Living Cells ◽  
Microtubule Depolymerization ◽  
Mathematical Simulations ◽  
Evolutionarily Conserved ◽  
Quantitative Examination ◽  
Insight Into

The kinetochore is a large, evolutionarily conserved protein structure that connects chromosomes with microtubules. During chromosome segregation, outer kinetochore components track depolymerizing ends of microtubules to facilitate the separation of chromosomes into two cells. In budding yeast, each chromosome has a point centromere upon which a single kinetochore is built, which attaches to a single microtubule. This defined architecture facilitates quantitative examination of kinetochores during the cell cycle. Using three independent measures—calibrated imaging, FRAP, and photoconversion—we find that the Dam1 submodule is unchanged during anaphase, whereas MIND and Ndc80 submodules add copies to form an “anaphase configuration” kinetochore. Microtubule depolymerization and kinesin-related motors contribute to copy addition. Mathematical simulations indicate that the addition of microtubule attachments could facilitate tracking during rapid microtubule depolymerization. We speculate that the minimal kinetochore configuration, which exists from G1 through metaphase, allows for correction of misattachments. Our study provides insight into dynamics and plasticity of the kinetochore structure during chromosome segregation in living cells.

Determination of protein stoichiometries via dual-color colocalization with single molecule localization microscopy

2021 ◽  
Author(s):  
Hua L Tan ◽  
Stefanie Bungert-Plümke ◽  
Daniel Kortzak ◽  
Christoph Fahlke ◽  
Gabriel Stölting
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Organic Anion ◽  
Protein Detection ◽  
Distribution Analysis ◽  
Localization Microscopy ◽  
Limited Volume ◽  
Dual Color ◽  
Single Molecule Localization Microscopy

The stoichiometry of plasma membrane protein complexes is an important determinant of their function and of interactions between the individual proteins. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine protein stoichiometries in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine protein stoichiometries, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual signals are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high detection efficiency for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the stoichiometry of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require strong temporal separation of emission events within the same diffraction-limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the stoichiometries of two SLC26 multifunctional anion exchangers and to determine the stoichiometries of four members of the SLC17 family of organic anion transporters.

scholarly journals Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

2018 ◽  
Vol 218 (2) ◽  
pp. 455-473 ◽  
Author(s):  
Cai Tong Ng ◽  
Li Deng ◽  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Depolymerization ◽  
Yeast Chromosome ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Electron Cryotomography ◽  
Binding Position

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

scholarly journals Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

2021 ◽  
Author(s):  
David Virant ◽  
Ilijana Vojnovic ◽  
Jannik Winkelmeier ◽  
Marc Endesfelder ◽  
Bartosz Turkowyd ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Single Molecule ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centromeric Chromatin ◽  
Localization Microscopy ◽  
Process Understanding ◽  
Segregation Process ◽  
Single Molecule Localization Microscopy

AbstractThe key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far.In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

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