scholarly journals Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

2006 ◽  
Vol 17 (3) ◽  
pp. 1239-1249 ◽  
Author(s):  
Joan C. Ritland Politz ◽  
Richard A. Tuft ◽  
Kannanganattu V. Prasanth ◽  
Nina Baudendistel ◽  
Kevin E. Fogarty ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Nuclear Bodies ◽  
Metabolic Energy ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Hybridization Probe ◽  
Fluorescence Correlation ◽  
Discernible Effect ◽  
Rate Of Movement

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

scholarly journals Fast and Reversible Photoswitching of the Fluorescent Protein Dronpa as Evidenced by Fluorescence Correlation Spectroscopy

2006 ◽  
Vol 91 (5) ◽  
pp. L45-L47 ◽  
Author(s):  
Peter Dedecker ◽  
Jun-ichi Hotta ◽  
Ryoko Ando ◽  
Atsushi Miyawaki ◽  
Yves Engelborghs ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1

2000 ◽  
Vol 113 (21) ◽  
pp. 3761-3768 ◽  
Author(s):  
I. Jagiello ◽  
A. Van Eynde ◽  
V. Vulsteke ◽  
M. Beullens ◽  
A. Boudrez ◽  
...  
Keyword(s):  
Active Transport ◽  
Protein Phosphatase ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Phosphatase 1 ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Nuclear Retention ◽  
Nuclear Localization Signals ◽  
Green Fluorescent

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy

2000 ◽  
Vol 113 (22) ◽  
pp. 3921-3930 ◽  
Author(s):  
R.H. Kohler ◽  
P. Schwille ◽  
W.W. Webb ◽  
M.R. Hanson
Keyword(s):  
Active Transport ◽  
Fluorescence Correlation Spectroscopy ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Long Distance ◽  
Fluorescence Correlation ◽  
Photon Excitation ◽  
Green Fluorescent

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

scholarly journals Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

2011 ◽  
Vol 22 (18) ◽  
pp. 3498-3507 ◽  
Author(s):  
Urszula Golebiewska ◽  
Jason G. Kay ◽  
Thomas Masters ◽  
Sergio Grinstein ◽  
Wonpil Im ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Surface Concentration ◽  
Correlation Spectroscopy ◽  
Similar Data ◽  
Fluorescence Correlation ◽  
The Many ◽  
Green Fluorescent

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

scholarly journals Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

2015 ◽  
Vol 26 (11) ◽  
pp. 2054-2066 ◽  
Author(s):  
Yinghua Guan ◽  
Matthias Meurer ◽  
Sarada Raghavan ◽  
Aleksander Rebane ◽  
Jake R. Lindquist ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescence Correlation Spectroscopy ◽  
Fluorescent Protein ◽  
Stokes Shift ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Quantitative Detection ◽  
Multiphoton Excitation ◽  
Fluorescence Correlation ◽  
Large Stokes Shift

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

Properties of Baculovirus Particles Displaying GFP Analyzed by Fluorescence Correlation Spectroscopy

2002 ◽  
Vol 383 (12) ◽  
pp. 1941-1946 ◽  
Author(s):  
J. Toivola ◽  
K. Ojala ◽  
P.O. Michel ◽  
M. Vuento ◽  
C. Oker-Blom
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Mean Value ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Fluorescence Correlation

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

scholarly journals Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

2001 ◽  
Vol 12 (11) ◽  
pp. 3502-3514 ◽  
Author(s):  
Marco Denegri ◽  
Ilaria Chiodi ◽  
Margherita Corioni ◽  
Fabio Cobianchi ◽  
Silvano Riva ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Bodies ◽  
Splicing Factors ◽  
Hnrnp A1 ◽  
Interacting Protein ◽  
Multifunctional Protein ◽  
Adenovirus E1a ◽  
Green Fluorescent ◽  
Nuclear Ribonucleoprotein ◽  
Processing Factors

Heterogeneous nuclear ribonucleoprotein (hnRNP) HAP (hnRNP A1 interacting protein) is a multifunctional protein with roles in RNA metabolism, transcription, and nuclear structure. After stress treatments, HAP is recruited to a small number of nuclear bodies, usually adjacent to the nucleoli, which consist of clusters of perichromatin granules and are depots of transcripts synthesized before stress. In this article we show that HAP bodies are sites of accumulation for a subset of RNA processing factors and are related to Sam68 nuclear bodies (SNBs) detectable in unstressed cells. Indeed, HAP and Sam68 are both present in SNBs and in HAP bodies, that we rename “stress-induced SNBs.” The determinants required for the redistribution of HAP lie between residue 580 and 788. Different portions of this region direct the recruitment of the green fluorescent protein to stress-induced SNBs, suggesting an interaction of HAP with different components of the bodies. With the use of the 580–725 region as bait in a two-hybrid screening, we have selected SRp30c and 9G8, two members of the SR family of splicing factors. Splicing factors are differentially affected by heat shock: SRp30c and SF2/ASF are efficiently recruited to stress-induced SNBs, whereas the distribution of SC35 is not perturbed. We propose that the differential sequestration of splicing factors could affect processing of specific transcripts. Accordingly, the formation of stress-induced SNBs is accompanied by a change in the splicing pattern of the adenovirus E1A transcripts.

scholarly journals Dynamics in HIV Gag Lattice Detected by Time-Lapse iPALM

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 65
Author(s):  
Ipsita Saha ◽  
Saveez Saffarian
Keyword(s):  
Lattice Dynamics ◽  
Fluorescent Protein ◽  
Function Analysis ◽  
Time Lapse ◽  
Correlation Spectroscopy ◽  
Virus Like Particles ◽  
Fluorescence Correlation ◽  
Hiv Virus ◽  
C Terminus ◽  
Fluorescent Tags

Immature HIV virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. This lattice has significant void spaces that were previously characterized by fluorescence correlation spectroscopy, cryoelectron tomography, and iPALM imaging. In the current study, we demonstrate that HIV virus-like particles (VLPs,) assembled by the viral protein Gag tagged at its C terminus with the photoactivable fluorescent protein Dendra, and are of the same size as virus-like particles assembled using only HIV Gag (140 ± 15 nm). We show that the Gag-Dendra lattice observed within these VLPs has similar gaps as those observed in Gag-only VLPs. We further used time-lapse iPALM microscopy to image the Gag–Dendra lattice within the lumen of VLPs at two timepoints. The reconstruction of these time-lapse images shows significant lattice dynamics within the lumen of purified VLPs. The addition of disuccinimidyl suberate (DSS) to the purified VLPs completely abrogated these dynamics. A method to quantify the dynamics of the Gag–Dendra lattice using correlation function analysis is further presented. The HIV Gag lattice, along with the structural lattices of many other viruses, have been mostly considered static. Our study provides an important tool to investigate the dynamics within these lattices and also highlights the effects of fluorescent tags on virion structures.

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