scholarly journals Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation

2018 ◽  
Author(s):  
Melissa R. Miller ◽  
Samuel J Kenny ◽  
Nadja Mannowetz ◽  
Steven A. Mansell ◽  
Michal Wojcik ◽  
...  
Keyword(s):  
Ion Channels ◽  
Regulatory Protein ◽  
Super Resolution ◽  
Human Sperm ◽  
Regulatory Proteins ◽  
Proton Channel ◽  
Structural Basis ◽  
Control Units ◽  
Proton Transporters ◽  
Super Resolution Microscopy

AbstractThe ability of sperm to fertilize an egg is controlled by ion channels, one of which is the pH-dependent calcium channel of sperm CatSper. For CatSper to be fully activated, the cytoplasmic pH must be alkaline, which is accomplished by either proton transporters, or a faster mechanism, such as the voltage-gated proton channel Hv1. To ensure effective regulation, these channels and regulatory proteins must be tightly compartmentalized. Here, we characterize human sperm nanodomains that are comprised of Hv1, CatSper and regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 forms asymmetrically positioned bilaterally distributed longitudinal lines that span the entire length of the sperm tail. Such a distribution provides a direct structural basis for the selective activation of CatSper, and subsequent flagellar rotation along the long axis that, together with hyperactivated motility, enhances sperm fertility. Indeed, Hv1 inhibition leads to a decrease in sperm rotation. Thus, sperm ion channels are organized in distinct regulatory nanodomains that control hyperactivated motility and rotation.

scholarly journals Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diogo Bessa-Neto ◽  
Gerti Beliu ◽  
Alexander Kuhlemann ◽  
Valeria Pecoraro ◽  
Sören Doose ◽  
...  
Keyword(s):  
Amino Acids ◽  
Regulatory Protein ◽  
Brain Slices ◽  
Transmembrane Proteins ◽  
Super Resolution ◽  
Bioorthogonal Labeling ◽  
Resolution Imaging ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy ◽  
Organotypic Brain Slices

AbstractProgress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

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>

Novel organic dyes for multicolor localization-based super-resolution microscopy

2015 ◽  
Vol 9 (1-2) ◽  
pp. 161-170 ◽  
Author(s):  
Martin Lehmann ◽  
Gregor Lichtner ◽  
Haider Klenz ◽  
Jan Schmoranzer
Keyword(s):  
Organic Dyes ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy

2021 ◽  
Author(s):  
Philipp Werther ◽  
Klaus Yserentant ◽  
Felix Braun ◽  
Kristin Grußmayer ◽  
Vytautas Navikas ◽  
...  
Keyword(s):  
Super Resolution ◽  
Live Cell ◽  
Fluorogenic Probes ◽  
Super Resolution Microscopy

scholarly journals Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Kristoffer Bernhem ◽  
Jacopo M. Fontana ◽  
Daniel Svensson ◽  
Liang Zhang ◽  
Linnéa M. Nilsson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chronic Diseases ◽  
Kidney Diseases ◽  
Super Resolution ◽  
Diabetic Patients ◽  
Diabetic Kidney ◽  
Renal Epithelial Cells ◽  
Apoptotic Process ◽  
Induced Apoptosis ◽  
Super Resolution Microscopy

AbstractActivation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

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