scholarly journals Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

Molecules ◽  
2014 ◽  
Vol 19 (8) ◽  
pp. 12116-12149 ◽  
Author(s):  
Beth Haas ◽  
Jyl Matson ◽  
Victor DiRita ◽  
Julie Biteen
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Nanometer Scale ◽  
Single Molecule Microscopy

scholarly journals Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol

2019 ◽  
Vol 116 (12) ◽  
pp. 5550-5557 ◽  
Author(s):  
Lucien E. Weiss ◽  
Ljiljana Milenkovic ◽  
Joshua Yoon ◽  
Tim Stearns ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Transmembrane Protein ◽  
Cellular Level ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Motional Dynamics ◽  
Directional Transport ◽  
Bulk Behavior

The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

scholarly journals A Photoactivatable Push−Pull Fluorophore for Single-Molecule Imaging in Live Cells

2008 ◽  
Vol 130 (29) ◽  
pp. 9204-9205 ◽  
Author(s):  
Samuel J. Lord ◽  
Nicholas R. Conley ◽  
Hsiao-lu D. Lee ◽  
Reichel Samuel ◽  
Na Liu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cells ◽  
Single Molecule Imaging

Single Molecule Height Measurements on a Membrane Protein in Nanometer-Scale Phospholipid Bilayer Disks

Langmuir ◽  
2000 ◽  
Vol 16 (14) ◽  
pp. 5993-5997 ◽  
Author(s):  
Timothy H. Bayburt ◽  
Joseph W. Carlson ◽  
Stephen G. Sligar
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Phospholipid Bilayer ◽  
Nanometer Scale

scholarly journals Combinefluent: An Open Source, Low-Cost Laser System for Single-Molecule Microscopy

2021 ◽  
Vol 120 (3) ◽  
pp. 184a
Author(s):  
Dylan George ◽  
Ashley Cadby ◽  
Timothy D. Craggs
Keyword(s):  
Open Source ◽  
Single Molecule ◽  
Low Cost ◽  
Laser System ◽  
Single Molecule Microscopy

Oblique-Sectional Single-Molecule Microscopy

2018 ◽  
Author(s):  
Jeongmin Kim ◽  
Michal Wojcik ◽  
Yuan Wang ◽  
Ke Xu ◽  
Xiang Zhang
Keyword(s):  
Single Molecule ◽  
Single Molecule Microscopy

scholarly journals Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

PLoS ONE ◽  
2017 ◽  
Vol 12 (11) ◽  
pp. e0188778 ◽  
Author(s):  
Yuki M. Shirai ◽  
Taka A. Tsunoyama ◽  
Nao Hiramoto-Yamaki ◽  
Koichiro M. Hirosawa ◽  
Akihiro C. E. Shibata ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Single Molecule Microscopy ◽  
Cortical Actin

scholarly journals Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging

2011 ◽  
Vol 192 (3) ◽  
pp. 463-480 ◽  
Author(s):  
Rinshi S. Kasai ◽  
Kenichi G. N. Suzuki ◽  
Eric R. Prossnitz ◽  
Ikuko Koyama-Honda ◽  
Chieko Nakada ◽  
...  
Keyword(s):  
Equilibrium Constant ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Dissociation Rate ◽  
Formyl Peptide Receptor ◽  
Live Cells ◽  
Imaging Method ◽  
Full Characterization ◽  
Before And After ◽  
Dissociation Rate Constants

Receptor dimerization is important for many signaling pathways. However, the monomer–dimer equilibrium has never been fully characterized for any receptor with a 2D equilibrium constant as well as association/dissociation rate constants (termed super-quantification). Here, we determined the dynamic equilibrium for the N-formyl peptide receptor (FPR), a chemoattractant G protein–coupled receptor (GPCR), in live cells at 37°C by developing a single fluorescent-molecule imaging method. Both before and after liganding, the dimer–monomer 2D equilibrium is unchanged, giving an equilibrium constant of 3.6 copies/µm2, with a dissociation and 2D association rate constant of 11.0 s−1 and 3.1 copies/µm2s−1, respectively. At physiological expression levels of ∼2.1 receptor copies/µm2 (∼6,000 copies/cell), monomers continually convert into dimers every 150 ms, dimers dissociate into monomers in 91 ms, and at any moment, 2,500 and 3,500 receptor molecules participate in transient dimers and monomers, respectively. Not only do FPR dimers fall apart rapidly, but FPR monomers also convert into dimers very quickly.

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