scholarly journals A quantitative map of nuclear pore assembly reveals two distinct mechanisms

2021 ◽  
Author(s):  
Shotaro Otsuka ◽  
Jeremy O.B. Tempkin ◽  
Antonio Z. Politi ◽  
Arina Rybina ◽  
M. Julius Hossain ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Computational Simulation ◽  
Correlation Spectroscopy ◽  
Nuclear Pore ◽  
Fluorescence Correlation ◽  
Molecular Events ◽  
Ring Complex ◽  
Dividing Cells ◽  
Nuclear Pore Assembly ◽  
Assembly Pathways

Understanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes. While we know that at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of their molecular events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. Our dynamic stoichiometry data allows us to perform the first computational simulation that predicts the structure of postmitotic NPC assembly intermediates.

The use of fluorescence correlation spectroscopy to characterise the molecular mobility of G protein-coupled receptors in membrane microdomains: an update

2021 ◽  
Vol 49 (4) ◽  
pp. 1547-1554
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Molecular Mechanisms ◽  
Short Review ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Membrane Microdomains ◽  
Sensitivity Threshold ◽  
Fluorescence Correlation ◽  
G Protein Coupled

It has become increasingly apparent that some G protein-coupled receptors (GPCRs) are not homogeneously expressed within the plasma membrane but may instead be organised within distinct signalling microdomains. These microdomains localise GPCRs in close proximity with other membrane proteins and intracellular signalling partners and could have profound implications for the spatial and temporal control of downstream signalling. In order to probe the molecular mechanisms that govern GPCR pharmacology within these domains, fluorescence techniques with effective single receptor sensitivity are required. Of these, fluorescence correlation spectroscopy (FCS) is a technique that meets this sensitivity threshold. This short review will provide an update of the recent uses of FCS based techniques in conjunction with GPCR subtype selective fluorescent ligands to characterise dynamic ligand–receptor interactions in whole cells and using purified GPCRs.

scholarly journals Postmitotic nuclear pore assembly proceeds by radial dilation of small ER membrane openings

2017 ◽  
Author(s):  
Shotaro Otsuka ◽  
Anna M. Steyer ◽  
Martin Schorb ◽  
Jean-Karim Hériché ◽  
M. Julius Hossain ◽  
...  
Keyword(s):  
Time Course ◽  
Three Dimensional ◽  
Membrane Topology ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Ring Complex ◽  
Pore Complexes ◽  
Transport Channels ◽  
Membrane Sealing ◽  
Nuclear Pore Assembly

AbstractThe nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

scholarly journals Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 802 ◽  
Author(s):  
Rumiana Tzoneva ◽  
Tihomira Stoyanova ◽  
Annett Petrich ◽  
Desislava Popova ◽  
Veselina Uzunova ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Cancer Cell ◽  
Fluorescence Correlation Spectroscopy ◽  
Lipid Membranes ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Generalized Polarization ◽  
Fluorescence Correlation

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid–lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Study of molecular events in cells by fluorescence correlation spectroscopy

2005 ◽  
Vol 62 (5) ◽  
pp. 535-550 ◽  
Author(s):  
V. Vukojević ◽  
A. Pramanik ◽  
T. Yakovleva ◽  
R. Rigler ◽  
L. Terenius ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Molecular Events ◽  
In Cells

scholarly journals Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models

2020 ◽  
Author(s):  
R. Tzoneva ◽  
T. Stoyanova ◽  
A. Petrich ◽  
D. Popova ◽  
A. Momchilova ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Cancer Cell ◽  
Fluorescence Correlation Spectroscopy ◽  
Lipid Membranes ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Generalized Polarization ◽  
Fluorescence Correlation

ABSTRACTAlkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, Erufosine (EPC3) is a promising drug for the treatment of several types of tumors which has not been sufficiently characterized. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy techniques (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy and generalized polarization imaging) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 might be linked to its effects on fundamental biophysical properties of lipid membranes in cancer cells.

scholarly journals Single mRNP analysis by super-resolution microscopy and fluorescence correlation spectroscopy reveals that small mRNP granules represent mRNA singletons

2019 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Frederik Otzen Bagger ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Translational Control ◽  
Fluorescence Correlation Spectroscopy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Nuclear Pore ◽  
Fluorescence Correlation ◽  
Super Resolution Microscopy

SummarySmall cytoplasmic mRNP granules are implicated in mRNA transport, translational control and decay. Employing Super-resolution Microscopy and Fluorescence Correlation Spectroscopy, we analyzed the molecular composition and dynamics of single cytoplasmic YBX1_IMP1 mRNP granules in live cells. Granules appeared elongated and branched with patches of IMP1 and YBX1 distributed along mRNA, reflecting the attachment of the two RNA-binding proteins in cis. Particles form at the nuclear pore and are spatially segregated from translating ribosomes, so the mRNP is a repository for mRNAs awaiting translation. In agreement with the average number of mRNA-binding sites derived from CLIP analyses, individual mRNPs contain 5 to 15 molecules of YBX1 and IMP1 and a single poly(A) tail identified by PABPC1. Taken together, we conclude that small cytoplasmic mRNP granules are mRNA singletons, thus depicting the cellular transcriptome. Consequently, expression of functionally related mRNAs in RNA regulons is unlikely to result from coordinated assembly.

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