scholarly journals The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

2020 ◽  
Vol 295 (42) ◽  
pp. 14279-14290 ◽  
Author(s):  
Avishek Prasai ◽  
Marketa Schmidt Cernohorska ◽  
Klara Ruppova ◽  
Veronika Niederlova ◽  
Monika Andelova ◽  
...  
Keyword(s):  
Cell Lines ◽  
Primary Cilia ◽  
Fluorescent Protein ◽  
Correlation Spectroscopy ◽  
Bardet Biedl Syndrome ◽  
Fluorescence Correlation ◽  
Biochemical Assays ◽  
Genes Encoding ◽  
Microscopy Techniques ◽  
Quantitative Fluorescence

Bardet–Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

scholarly journals The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells

2020 ◽  
Author(s):  
Avishek Prasai ◽  
Marketa Schmidt Cernohorska ◽  
Klara Ruppova ◽  
Veronika Niederlova ◽  
Monika Andelova ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Primary Cilia ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Comprehensive Analysis ◽  
Sequential Process ◽  
Bardet Biedl Syndrome ◽  
Epithelial Cell Lines ◽  
Genes Encoding

AbstractBardet-Biedl Syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. Most BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, BBSome, which mediates the trafficking of ciliary cargoes. Although, the structure of the BBSome has been resolved recently, the mechanism of assembly of this complicated complex in living cells is poorly understood. We generated a large library of human retinal epithelial cell lines deficient in particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We performed a comprehensive analysis of these cell lines using biochemical and microscopy approaches. Our data revealed that the BBSome formation is a sequential process including a step of the pre-BBSome assembly at pericentriolar satellites nucleated by BBS4, followed by the translocation of the BBSome into the ciliary base mediated by BBS1.

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

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 Nuclear pores as versatile reference standards for quantitative superresolution microscopy

2019 ◽  
Author(s):  
Jervis Vermal Thevathasan ◽  
Maurice Kahnwald ◽  
Konstanty Cieśliński ◽  
Philipp Hoess ◽  
Sudheer Kumar Peneti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Fluorescent Protein ◽  
Reference Standards ◽  
Nuclear Pore ◽  
Biologically Relevant ◽  
Superresolution Microscopy ◽  
Absolute Measurements ◽  
Quantitative Fluorescence

AbstractQuantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.Here we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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.

Sign in / Sign up

Export Citation Format

Share Document