scholarly journals Photobleaching step analysis for robust determination of protein complex stoichiometries

2020 ◽  
Author(s):  
Johan Hummert ◽  
Klaus Yserentant ◽  
Theresa Fink ◽  
Jonas Euchner ◽  
Dirk-Peter Herten
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Computational Cost ◽  
Cellular Structures ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Single Molecule Level ◽  
Copy Numbers ◽  
Counting Range ◽  
Pore Complexes

AbstractThe composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labeled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

scholarly journals Photobleaching step analysis for robust determination of protein complex stoichiometries

2021 ◽  
Author(s):  
Johan Hummert ◽  
Klaus Yserentant ◽  
Theresa Fink ◽  
Jonas Euchner ◽  
Yin Xin Ho ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Complex ◽  
Computational Cost ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Single Molecule Level ◽  
High Data ◽  
Counting Range ◽  
Pore Complexes

The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high data quality. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

scholarly journals Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus

2016 ◽  
Vol 27 (17) ◽  
pp. 2742-2756 ◽  
Author(s):  
Biplab Paul ◽  
Ben Montpetit
Keyword(s):  
Single Molecule ◽  
Rna Processing ◽  
Nuclear Export ◽  
Essential Gene ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Eukaryotic Gene ◽  
Transcription Sites ◽  
Pore Complexes

Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

scholarly journals Multidomain ribosomal protein trees and the planctobacterial origin of neomura (eukaryotes, archaebacteria)

PROTOPLASMA ◽  
2020 ◽  
Vol 257 (3) ◽  
pp. 621-753 ◽  
Author(s):  
Thomas Cavalier-Smith ◽  
Ema E-Yung Chao
Keyword(s):  
Maximum Likelihood ◽  
Ribosomal Protein ◽  
Outer Membrane ◽  
Cell Biology ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Wall Losses ◽  
Historical Pattern ◽  
Pore Complexes ◽  
Intracellular Motility

AbstractPalaeontologically, eubacteria are > 3× older than neomura (eukaryotes, archaebacteria). Cell biology contrasts ancestral eubacterial murein peptidoglycan walls and derived neomuran N-linked glycoprotein coats/walls. Misinterpreting long stems connecting clade neomura to eubacteria on ribosomal sequence trees (plus misinterpreted protein paralogue trees) obscured this historical pattern. Universal multiprotein ribosomal protein (RP) trees, more accurate than rRNA trees, are taxonomically undersampled. To reduce contradictions with genically richer eukaryote trees and improve eubacterial phylogeny, we constructed site-heterogeneous and maximum-likelihood universal three-domain, two-domain, and single-domain trees for 143 eukaryotes (branching now congruent with 187-protein trees), 60 archaebacteria, and 151 taxonomically representative eubacteria, using 51 and 26 RPs. Site-heterogeneous trees greatly improve eubacterial phylogeny and higher classification, e.g. showing gracilicute monophyly, that many ‘rDNA-phyla’ belong in Proteobacteria, and reveal robust new phyla Synthermota and Aquithermota. Monoderm Posibacteria and Mollicutes (two separate wall losses) are both polyphyletic: multiple outer membrane losses in Endobacteria occurred separately from Actinobacteria; neither phylum is related to Chloroflexi, the most divergent prokaryotes, which originated photosynthesis (new model proposed). RP trees support an eozoan root for eukaryotes and are consistent with archaebacteria being their sisters and rooted between Filarchaeota (=Proteoarchaeota, including ‘Asgardia’) and Euryarchaeota sensu-lato (including ultrasimplified ‘DPANN’ whose long branches often distort trees). Two-domain trees group eukaryotes within Planctobacteria, and archaebacteria with Planctobacteria/Sphingobacteria. Integrated molecular/palaeontological evidence favours negibacterial ancestors for neomura and all life. Unique presence of key pre-neomuran characters favours Planctobacteria only as ancestral to neomura, which apparently arose by coevolutionary repercussions (explained here in detail, including RP replacement) of simultaneous outer membrane and murein loss. Planctobacterial C-1 methanotrophic enzymes are likely ancestral to archaebacterial methanogenesis and β-propeller-α-solenoid proteins to eukaryotic vesicle coats, nuclear-pore-complexes, and intraciliary transport. Planctobacterial chaperone-independent 4/5-protofilament microtubules and MamK actin-ancestors prepared for eukaryote intracellular motility, mitosis, cytokinesis, and phagocytosis. We refute numerous wrong ideas about the universal tree.

scholarly journals Nuclear import time and transport efficiency depend on importin β concentration

2006 ◽  
Vol 174 (7) ◽  
pp. 951-961 ◽  
Author(s):  
Weidong Yang ◽  
Siegfried M. Musser
Keyword(s):  
Single Molecule ◽  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transport Efficiency ◽  
Bidirectional Transport ◽  
Pore Complexes ◽  
Transit Speed ◽  
And Control

Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions with NPCs. At low importin β concentrations, about half of the signal-dependent cargos that interacted with an NPC were translocated across the NE, indicating a nuclear import efficiency of ∼50%. At high importin β concentrations, the import efficiency increased to ∼80% and the transit speed increased approximately sevenfold. The transit speed and import efficiency of a signal-independent cargo was also increased by high importin β concentrations. These results demonstrate that maximum nucleocytoplasmic transport velocities can be modulated by at least ∼10-fold by the importin β concentration and therefore suggest a potential mechanism for regulating the speed of cargo traffic across the NE.

scholarly journals Detecting structural heterogeneity in single-molecule localization microscopy data

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Teun A.P.M. Huijben ◽  
Hamidreza Heydarian ◽  
Alexander Auer ◽  
Florian Schueder ◽  
Ralf Jungmann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
A Priori ◽  
Unsupervised Classification ◽  
Structural Heterogeneity ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Localization Microscopy ◽  
Pore Complexes ◽  
Single Molecule Localization Microscopy

AbstractParticle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity metric. We achieve 96% classification accuracy on mixtures of up to four different DNA-origami structures, detect rare classes of origami occuring at 2% rate, and capture variation in ellipticity of nuclear pore complexes.

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