scholarly journals Analysis of novel hyperosmotic shock response suggests “beads in liquid” cytosol structure

2019 ◽  
Author(s):  
A.I. Alexandrov ◽  
E.V. Grosfeld ◽  
A.A. Dergalev ◽  
V.V. Kushnirov ◽  
R.N. Chuprov-Netochin ◽  
...  
Keyword(s):  
Super Resolution ◽  
Close Correlation ◽  
Shock Onset ◽  
Hyperosmotic Shock ◽  
Amyloidogenic Proteins ◽  
Genome Wide ◽  
A Genome ◽  
Targeted Testing ◽  
Body Components ◽  
Super Resolution Microscopy

AbstractProteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we propose a new model of the cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

scholarly journals Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Junsheng Yang ◽  
Xinxin Hao ◽  
Xiuling Cao ◽  
Beidong Liu ◽  
Thomas Nyström
Keyword(s):  
Quality Control ◽  
Regulatory System ◽  
Super Resolution ◽  
Age Related ◽  
Genome Wide ◽  
A Genome ◽  
Polyglutamine Expansions ◽  
Key Factor ◽  
Super Resolution Microscopy ◽  
Control Complex

Huntington disease (HD) is a neurological disorder caused by polyglutamine expansions in mutated Huntingtin (mHtt) proteins, rendering them prone to form inclusion bodies (IB). We report that in yeast, such IB formation is a factor-dependent process subjected to age-related decline. A genome-wide, high-content imaging approach, identified the E3 ubiquitin ligase, Ltn1 of the ribosome quality control complex (RQC) as a key factor required for IB formation, ubiquitination, and detoxification of model mHtt. The failure of ltn1∆ cells to manage mHtt was traced to another RQC component, Tae2, and inappropriate control of heat shock transcription factor, Hsf1, activity. Moreover, super-resolution microscopy revealed that mHtt toxicity in RQC-deficient cells was accompanied by multiple mHtt aggregates altering actin cytoskeletal structures and retarding endocytosis. The data demonstrates that spatial sequestration of mHtt into IBs is policed by the RQC-Hsf1 regulatory system and that such compartmentalization, rather than ubiquitination, is key to mHtt detoxification.

scholarly journals Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei

2019 ◽  
Vol 295 (3) ◽  
pp. 729-742
Author(s):  
Kieu T. M. Pham ◽  
Ziyin Li
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Super Resolution ◽  
Specific Protein ◽  
Protein Stabilization ◽  
Structured Illumination ◽  
Human Parasite ◽  
Evolutionarily Conserved ◽  
Body Components ◽  
Super Resolution Microscopy

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

scholarly journals Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations

2021 ◽  
Author(s):  
Michal Franek ◽  
Agata Kilar ◽  
Petr Fojtík ◽  
Marie Olšinová ◽  
Aleš Benda ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Super Resolution ◽  
Single Copy ◽  
Histone Variants ◽  
Dna Repeats ◽  
Genome Wide ◽  
Custom Script ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Super Resolution Microscopy ◽  
Dna Methylation Analysis

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. While uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques based on averaging cell populations or high number of repeats in a single cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context and thus constitutes an important tool for a wholesome understanding of the epigenetic organization of genomes. We present that using an optimized fiber extension protocol is essential to obtain more reproducible data, where the clustering of fibers is minimized. We also demonstrate that applying super-resolution microscopy is important to reliably evaluate the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script to analyse methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

scholarly journals A Genome-Wide Transcription Analysis Reveals a Close Correlation of Promoter INDEL Polymorphism and Heterotic Gene Expression in Rice Hybrids

2008 ◽  
Vol 1 (5) ◽  
pp. 720-731 ◽  
Author(s):  
Hui-Yong Zhang ◽  
Hang He ◽  
Liang-Bi Chen ◽  
Lei Li ◽  
Man-Zhong Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Close Correlation ◽  
Indel Polymorphism ◽  
Transcription Analysis ◽  
Genome Wide ◽  
A Genome

scholarly journals Periodic actin structures in neuronal axons are required to maintain microtubules

2016 ◽  
Author(s):  
Yue Qu ◽  
Ines Hahn ◽  
Stephen Webb ◽  
Simon P. Pearce ◽  
Andreas Prokop
Keyword(s):  
Super Resolution ◽  
Close Correlation ◽  
Membrane Skeleton ◽  
Cortical Actin ◽  
Evolutionarily Conserved ◽  
Genes Encoding ◽  
Neuronal Processes ◽  
And Function ◽  
Super Resolution Microscopy ◽  
Actin Rings

SummaryAxons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration.Significance statementAxons are cable-like neuronal processes that are up to a meter long in humans. These delicate structures often need to be maintained for an organism’s lifetime, i.e. up to a century in humans. Unsurprisingly, we gradually lose about 50% of axons as we age. Bundles of microtubules form the structural backbones and highways for life-sustaining transport within axons, and maintenance of these bundles is essential for axonal longevity. However, the mechanisms which actively maintain axonal microtubules are poorly understood. Here we identify cortical actin as an important factor maintaining microtubule polymerisation in axons. This finding provides potential explanations for the previously identified, but unexplained, links between mutations in genes encoding cortical actin regulators and neurodegeneration.

scholarly journals aCPSF1 controlled archaeal transcription termination: a prototypical eukaryotic model

2019 ◽  
Author(s):  
Lei Yue ◽  
Jie Li ◽  
Bing Zhang ◽  
Lei Qi ◽  
Fangqing Zhao ◽  
...  
Keyword(s):  
Transcription Termination ◽  
Super Resolution ◽  
Microscopy Imaging ◽  
Genome Wide ◽  
Transcription Termination Factor ◽  
A Genome ◽  
Cold Sensitive ◽  
Archaeal Transcription ◽  
Rnap Ii

AbstractTranscription termination defines RNA 3′-ends and guarantees programmed transcriptomes, thus is an essential biological process for life. However, transcription termination mechanisms remain almost unknown in Archaea. Here reported the first general transcription termination factor of Archaea, the conserved ribonuclease aCPSF1, and elucidated its 3′-end cleavage dependent termination mechanism. Depletion of Mmp-aCPSF1 in a methanoarchaeon Methanococcus maripaludis caused a genome-wide transcription termination defect and overall transcriptome chaos, and cold-sensitive growth. Transcript-3′end-sequencing (Term-seq) revealed transcriptions mostly terminated downstream of a uridine-rich terminator motif, where Mmp-aCPSF1 performed cleavage. The endoribonuclease activity was determined essential to terminate transcription in vivo as well. Through super-resolution photoactivated localization microscopy imaging, co-immunoprecipitation, and chromatin immunoprecipitation, we demonstrated that Mmp-aCPSF1 localizes within nucleoid and associates with RNAP and chromosomes. aCPSF1 appears to co-evolve with archaeal RNAPs, and two distant orthologs each from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to termination transcription. Thus, aCPSF1 dependent termination mechanism could be universally employed in Archaea, including Lokiarchaeota, one supposed archaeal ancestor of Eukaryotes. Therefore, the reported aCPSF1 cleavage-dependent termination mode not only hints an archetype of Eukaryotic 3′-end processing/cleavage triggered RNAP II termination, but also would shed lights on understanding the complex eukaryotic termination based on the simplified archaeal model.

scholarly journals Stochastic association of neighboring replicons creates replication factories in budding yeast

2013 ◽  
Vol 202 (7) ◽  
pp. 1001-1012 ◽  
Author(s):  
Nazan Saner ◽  
Jens Karschau ◽  
Toyoaki Natsume ◽  
Marek Gierliński ◽  
Renata Retkute ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Super Resolution ◽  
Replication Proteins ◽  
Replication Factory ◽  
Genome Wide ◽  
Nuclear Structures ◽  
Super Resolution Microscopy ◽  
Insight Into

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy
Sign in / Sign up

Export Citation Format

Share Document