scholarly journals A mitotic screen in Indian muntjac cells unveils how Augmin drives kinetochore fiber maturation

2021 ◽  
Author(s):  
Ana C Almeida ◽  
Joana Oliveira ◽  
Danica Drpic ◽  
Liam Cheeseman ◽  
Joana Damas ◽  
...  
Keyword(s):  
Chromosome Number ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Super Resolution ◽  
Fundamental Question ◽  
Angular Dispersion ◽  
Indian Muntjac ◽  
Main Driver ◽  
Microtubule Growth ◽  
Functional Analyses

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fibers. How k-fibers mature from initial kinetochore-microtubule attachments remains a fundamental question. Here we used the low chromosome number (n=3) and distinctively large kinetochores of Indian muntjac cells to investigate the molecular mechanism underlying k-fiber maturation. By combining functional analyses of 64 conserved mitotic proteins with fixed- and live-cell super-resolution CH-STED nanoscopy, we identified Augmin as the main driver of k-fiber maturation. Augmin promoted kinetochore microtubule turnover by sustaining centrosome-independent microtubule growth from kinetochores and poleward flux. Tracking of microtubule growth events in the kinetochore vicinity revealed a wide angular dispersion, consistent with Augmin-mediated branched microtubule nucleation. Indeed, Augmin depletion reduced the frequency of microtubule growth events on individual k-fibers and prevented normal repair after acute k-fiber injury by laser microsurgery. Altogether, our work directly elucidates how Augmin mediates k-fiber maturation in mammals.

scholarly journals Chromosome segregation is driven by joint microtubule sliding action of kinesins KIF4A and EG5

2019 ◽  
Author(s):  
Kruno Vukušić ◽  
Renata Buđa ◽  
Ivana Ponjavić ◽  
Patrik Risteski ◽  
Iva M. Tolić
Keyword(s):  
Cell Division ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Super Resolution ◽  
Human Cells ◽  
Microtubule Stability ◽  
Sliding Mechanism ◽  
Spindle Elongation ◽  
Super Resolution Microscopy

Successful cell division requires proper chromosome segregation during anaphase. Forces required for chromosome segregation in human cells are linked to sliding of antiparallel microtubules and sliding capacity has been demonstrated in vitro for multiple motor proteins, but the molecular mechanism of sliding in the spindle of human cells remains unknown. Using combined depletion and inactivation assays to explore redundancy between multiple targets together with CRISPR technology, we found that PRC1-dependent motor KIF4A/kinesin-4, together with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Photoactivation of tubulin and super-resolution microscopy show that perturbation of both proteins impairs sliding, while decreased midzone microtubule stability cannot explain the observed anaphase arrest. Thus, two independent sliding modules power sliding mechanism that drives spindle elongation in human cells.

scholarly journals Structural and functional conservation of the programmed −1 ribosomal frameshift signal of SARS-CoV-2

2020 ◽  
Author(s):  
Jamie A. Kelly ◽  
Alexandra N. Olson ◽  
Krishna Neupane ◽  
Sneha Munshi ◽  
Josue San Emeterio ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Small Molecule ◽  
Ribosomal Frameshift ◽  
Short Term ◽  
X Ray ◽  
Functional Conservation ◽  
Lead Compounds ◽  
X Ray Scattering ◽  
The World ◽  
Functional Analyses

Abstract17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called −1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of −1 PRF, and that it also harbors a −1 PRF attenuation element. Altering −1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of −1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated −1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit −1 PRF was similarly effective against −1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

COVID-19 Shock and Schumpeterian “Creative Destruction”

2021 ◽  
pp. 272-313
Keyword(s):  
Industrial Revolution ◽  
Self Regulation ◽  
Creative Destruction ◽  
Fundamental Question ◽  
Dynamic State ◽  
Dynamic Processes ◽  
Private Business ◽  
Self Development ◽  
Main Driver ◽  
Main Factor

The COVID-19 pandemic shock pushed the objective processes of discontinuity in the evolution of a static economy. The new path of self-development of the economy in its dynamic state starts with transferring system to the basis of the Industrial Revolution 4.0. In this connection, the fundamental question arises related to what structure will be the main driver of the future dynamic processes. In this regard, the idea of J. Schumpeter about the entrepreneurs' ability to carry out “creative destruction” becomes highly relevant. It is about private business capable of self-regulation as well as building post-coronavirus systemic integrities both in the technological sphere, in society and economy. It is about their forced by COVID-19 understanding of the main factor of their success, associated with the employees and their potential which is largely formed in society and used in firms if they construct their internal organization on the principles of human centrism.

scholarly journals The stoichiometry of the outer kinetochore is modulated by microtubule-proximal regulatory factors

2019 ◽  
Vol 218 (7) ◽  
pp. 2124-2135 ◽  
Author(s):  
Karthik Dhatchinamoorthy ◽  
Jay R. Unruh ◽  
Jeffrey J. Lange ◽  
Michaella Levy ◽  
Brian D. Slaughter ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Particle ◽  
Super Resolution ◽  
Molecular Machine ◽  
Cyclin Dependent Kinase ◽  
Centromeric Dna ◽  
Microtubule Binding ◽  
Regulatory Factors

The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule–kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore–microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation.

scholarly journals Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes

2019 ◽  
pp. 267-295 ◽  
Author(s):  
Binyam Mogessie
Keyword(s):  
Functional Analysis ◽  
High Resolution ◽  
Chromosome Segregation ◽  
Actin Filaments ◽  
Immunofluorescence Microscopy ◽  
Super Resolution ◽  
Oocyte Meiosis ◽  
Meiotic Spindles

Abstract Chromosome segregation is conserved throughout eukaryotes. In most systems, it is solely driven by a spindle machinery that is assembled from microtubules. We have recently discovered that actin filaments that are embedded inside meiotic spindles (spindle actin) are needed for accurate chromosome segregation in mammalian oocytes. To understand the function of spindle actin in oocyte meiosis, we have developed high-resolution and super-resolution live and immunofluorescence microscopy assays that are described in this chapter.

scholarly journals The size of the EB cap determines instantaneous microtubule stability

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Christian Duellberg ◽  
Nicholas I Cade ◽  
David Holmes ◽  
Thomas Surrey
Keyword(s):  
Molecular Mechanism ◽  
Quantitative Model ◽  
Kinetic Characteristics ◽  
Tirf Microscopy ◽  
Short Region ◽  
Microtubule Stability ◽  
Binding Regions ◽  
Microtubule Growth ◽  
Insight Into

The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.

scholarly journals Proteomic and functional analyses of the periodic membrane skeleton in neurons

2020 ◽  
Author(s):  
Ruobo Zhou ◽  
Boran Han ◽  
Roberta Nowak ◽  
Yunzhe Lu ◽  
Evan Heller ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Super Resolution ◽  
Membrane Skeleton ◽  
Interacting Proteins ◽  
Functional Roles ◽  
Genetic Perturbations ◽  
Resolution Imaging ◽  
Functional Analyses

AbstractActin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of candidate MPS-interacting proteins that span diverse functional categories. We validated representative proteins in several of these categories, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, demonstrating periodic distributions of ∼20 proteins in neurons using super-resolution imaging. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide new insights into the interactome of the MPS, and suggest new functions of the MPS in neurons.

scholarly journals A pipeline for multidimensional confocal analysis of mitochondrial morphology, function and dynamics in pancreatic β-cells

2019 ◽  
Author(s):  
Ahsen Chaudhry ◽  
Rocky Shi ◽  
Dan S. Luciani
Keyword(s):  
Metabolic Diseases ◽  
Super Resolution ◽  
Cell Types ◽  
Mitochondrial Morphology ◽  
State Function ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Health And Disease ◽  
Mitochondrial Networks ◽  
Functional Analyses

ABSTRACTLive-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software it is challenging to accurately extract features from dense multi-layered mitochondrial networks, such as those in insulin-secreting pancreatic β-cells. Motivated by this, we developed a comprehensive pipeline, and associated ImageJ plugin, that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse β-cells, and by extension other similarly challenging cell-types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photo-labelling and unsupervised cluster analysis, and is capable of morphological and functional analyses on a per-organelle basis, including in 4D (xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function, and provides a valuable resource to accelerate mitochondrial research in health and disease.

scholarly journals A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells

2020 ◽  
Vol 318 (2) ◽  
pp. E87-E101 ◽  
Author(s):  
Ahsen Chaudhry ◽  
Rocky Shi ◽  
Dan S. Luciani
Keyword(s):  
Metabolic Diseases ◽  
Super Resolution ◽  
Cell Types ◽  
Mitochondrial Morphology ◽  
State Function ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Health And Disease ◽  
Mitochondrial Networks ◽  
Functional Analyses

Live-cell imaging of mitochondrial function and dynamics can provide vital insights into both physiology and pathophysiology, including of metabolic diseases like type 2 diabetes. However, without super-resolution microscopy and commercial analysis software, it is challenging to accurately extract features from dense multilayered mitochondrial networks, such as those in insulin-secreting pancreatic β-cells. Motivated by this, we developed a comprehensive pipeline and associated ImageJ plugin that enables 2D/3D quantification of mitochondrial network morphology and dynamics in mouse β-cells and by extension other similarly challenging cell types. The approach is based on standard confocal microscopy and shareware, making it widely accessible. The pipeline was validated using mitochondrial photolabeling and unsupervised cluster analysis and is capable of morphological and functional analyses on a per-organelle basis, including in 4D ( xyzt). Overall, this tool offers a powerful framework for multiplexed analysis of mitochondrial state/function and provides a valuable resource to accelerate mitochondrial research in health and disease.

scholarly journals Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

2011 ◽  
Vol 22 (19) ◽  
pp. 3584-3594 ◽  
Author(s):  
Randy Wei ◽  
Bryan Ngo ◽  
Guikai Wu ◽  
Wen-Hwa Lee
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Distribution Characteristic ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Ndc80 Complex ◽  
Complex Protein

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

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