scholarly journals CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Michael Ratz ◽  
Ilaria Testa ◽  
Stefan W. Hell ◽  
Stefan Jakobs
Keyword(s):  
Super Resolution ◽  
Human Cells ◽  
Endogenous Protein ◽  
Super Resolution Microscopy ◽  
Protein Tagging

scholarly journals Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies

ACS Nano ◽  
2015 ◽  
Vol 9 (11) ◽  
pp. 11034-11041 ◽  
Author(s):  
Ingrid C. Vreja ◽  
Ivana Nikić ◽  
Fabian Göttfert ◽  
Mark Bates ◽  
Katharina Kröhnert ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Protein Assemblies ◽  
Super Resolution Microscopy ◽  
Protein Tagging

scholarly journals Chromosome segregation is driven by joint microtubule sliding action of kinesins KIF4A and EG5

2019 ◽  
Author(s):  
Kruno Vukušić ◽  
Renata Buđa ◽  
Ivana Ponjavić ◽  
Patrik Risteski ◽  
Iva M. Tolić
Keyword(s):  
Cell Division ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Super Resolution ◽  
Human Cells ◽  
Microtubule Stability ◽  
Sliding Mechanism ◽  
Spindle Elongation ◽  
Super Resolution Microscopy

Successful cell division requires proper chromosome segregation during anaphase. Forces required for chromosome segregation in human cells are linked to sliding of antiparallel microtubules and sliding capacity has been demonstrated in vitro for multiple motor proteins, but the molecular mechanism of sliding in the spindle of human cells remains unknown. Using combined depletion and inactivation assays to explore redundancy between multiple targets together with CRISPR technology, we found that PRC1-dependent motor KIF4A/kinesin-4, together with EG5/kinesin-5 motor is essential for spindle elongation in human cells. Photoactivation of tubulin and super-resolution microscopy show that perturbation of both proteins impairs sliding, while decreased midzone microtubule stability cannot explain the observed anaphase arrest. Thus, two independent sliding modules power sliding mechanism that drives spindle elongation in human cells.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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