scholarly journals Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

2019 ◽  
Vol 47 (4) ◽  
pp. 1117-1129 ◽  
Author(s):  
David G. Priest ◽  
Ashleigh Solano ◽  
Jieqiong Lou ◽  
Elizabeth Hinde
Keyword(s):  
Nuclear Protein ◽  
Nuclear Architecture ◽  
Nuclear Proteins ◽  
Protein Diffusion ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Fluorescent Tracer ◽  
Target Search ◽  
Chromatin Compaction ◽  
Structural Framework ◽  
The Impact

Abstract Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

scholarly journals Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giulia Tedeschi ◽  
Lorenzo Scipioni ◽  
Maria Papanikolaou ◽  
Geoffrey W. Abbott ◽  
Michelle A. Digman
Keyword(s):  
Plasma Membrane ◽  
Protein Diffusion ◽  
Ion Diffusion ◽  
Fluorescence Fluctuation Spectroscopy ◽  
Biophysical Characterization ◽  
Kv Channels ◽  
Subunit Stoichiometry ◽  
Spatio Temporal ◽  
Voltage Sensing Domain

AbstractVoltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

Evolutionary origin of the cell nucleus and its functional architecture

2010 ◽  
Vol 48 ◽  
pp. 1-24 ◽  
Author(s):  
Jan Postberg ◽  
Hans J. Lipps ◽  
Thomas Cremer
Keyword(s):  
Cell Nucleus ◽  
Interdisciplinary Research ◽  
Nuclear Organization ◽  
Nuclear Architecture ◽  
Evolutionary Origin ◽  
Cell Type ◽  
Research Strategies ◽  
Evolutionarily Conserved ◽  
Species Specific ◽  
The Impact

Understanding the evolutionary origin of the nucleus and its compartmentalized architecture provides a huge but, as expected, greatly rewarding challenge in the post-genomic era. We start this chapter with a survey of current hypotheses on the evolutionary origin of the cell nucleus. Thereafter, we provide an overview of evolutionarily conserved features of chromatin organization and arrangements, as well as topographical aspects of DNA replication and transcription, followed by a brief introduction of current models of nuclear architecture. In addition to features which may possibly apply to all eukaryotes, the evolutionary plasticity of higher-order nuclear organization is reflected by cell-type- and species-specific features, by the ability of nuclear architecture to adapt to specific environmental demands, as well as by the impact of aberrant nuclear organization on senescence and human disease. We conclude this chapter with a reflection on the necessity of interdisciplinary research strategies to map epigenomes in space and time.

scholarly journals Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627 ◽  
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

scholarly journals The Impact of Nuclear Architecture on EGFP Diffusion Revealed by Pair Correlation Analysis

2011 ◽  
Vol 100 (3) ◽  
pp. 67a ◽  
Author(s):  
Elizabeth Hinde ◽  
Francesco Cardarelli ◽  
Michelle A. Digman ◽  
Enrico Gratton
Keyword(s):  
Correlation Analysis ◽  
Pair Correlation ◽  
Nuclear Architecture ◽  
The Impact

scholarly journals PROTEINS IN NUCLEOCYTOPLASMIC INTERACTIONS

1968 ◽  
Vol 39 (2) ◽  
pp. 404-414 ◽  
Author(s):  
David Prescott ◽  
Lester Goldstein
Keyword(s):  
Binding Sites ◽  
Interphase Nucleus ◽  
Nuclear Protein ◽  
Protein A ◽  
Amoeba Proteus ◽  
Nuclear Proteins ◽  
Nuclear Transplantation ◽  
Amino Acid Labeling ◽  
Turn Over ◽  
Nucleocytoplasmic Interactions

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.

scholarly journals In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.

1986 ◽  
Vol 103 (6) ◽  
pp. 2091-2102 ◽  
Author(s):  
D D Newmeyer ◽  
D R Finlay ◽  
D J Forbes
Keyword(s):  
Low Temperature ◽  
Nuclear Protein ◽  
Transport Model ◽  
Nuclear Transport ◽  
Atp Depletion ◽  
Nuclear Proteins ◽  
Direct Role ◽  
In Vitro System

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy-dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Bürglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non-nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.

IMMUNOFLUORESCENT STUDIES OF BASIC NUCLEAR PROTEINS

1970 ◽  
Vol 12 (3) ◽  
pp. 570-581 ◽  
Author(s):  
L. A. David ◽  
Margaret J. Corey
Keyword(s):  
Nuclear Protein ◽  
Polytene Chromosomes ◽  
Nuclear Proteins ◽  
Root Tip ◽  
Salivary Gland Cells ◽  
Blood Smears ◽  
Calf Thymus ◽  
Species Specific ◽  
Phylogenetic Divergence ◽  
Tip Cells

Histone antibodies were obtained by injecting salt extracted calf thymus basic nuclear proteins into chickens. Fluorescent globulins were prepared from chicken antisera with precipitating antibodies against a single electrophoretic fraction of the nuclear protein extract. The fluorescent antiserum was specific for nuclei and chromosomes but was neither organ nor species specific. It reacted specifically with nuclei of cell imprints of bovine and human leucocytes, with polytene chromosomes from Chironomus salivary gland cells, and with nuclei from lily and onion root tip cells. It did not react with chicken blood smears. A decrease in the intensity of fluorescence roughly proportional to the phylogenetic divergence of the tested species was observed.These studies indicate that this basic nuclear protein fraction has acquired remarkably little variation over a long evolutionary period.

scholarly journals Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

2020 ◽  
Author(s):  
Mathew Stracy ◽  
Jakob Schweizer ◽  
David J Sherratt ◽  
Achillefs N Kapanidis ◽  
Stephan Uphoff ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Search Time ◽  
Dna Binding Proteins ◽  
Protein Diffusion ◽  
Chromosomal Dna ◽  
Dna Interactions ◽  
Target Search ◽  
Molecular Weights

ABSTRACTDespite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

scholarly journals Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

2020 ◽  
Author(s):  
Colin Hockings ◽  
Chetan Poudel ◽  
Kevin A. Feeney ◽  
Clara L. Novo ◽  
Mehdi S. Hamouda ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Quantitative Method ◽  
Embryonic Stem ◽  
Nuclear Architecture ◽  
Live Cells ◽  
Fixed Tissue ◽  
Chromatin Compaction ◽  
Tissue Sections ◽  
Cell Transition ◽  
Live Cancer Cell

The global compaction state of chromatin in a nucleus is an important component of cell identity that has been difficult to measure. We have developed a quantitative method to measure the chromatin compaction state in both live and fixed cells, without the need for genetic modification, using the fluorescence lifetime of SiR-DNA dye. After optimising this method using live cancer cell lines treated to induce chromatin compaction or decompaction, we observed chromatin compaction in differentiating epithelial cells in fixed tissue sections, as well as local decompaction foci that may represent transcription factories. In addition, we shed new light on chromatin decompaction during embryonic stem cell transition out of their naïve pluripotent state. This method will be useful to studies of nuclear architecture, and may be easy, cheap, and accessible enough to serve as a general assay of ‘stem-ness’.

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