scholarly journals Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Patrina A Pellett ◽  
Felix Dietrich ◽  
Jörg Bewersdorf ◽  
James E Rothman ◽  
Grégory Lavieu
Keyword(s):  
Steady State ◽  
Transport Model ◽  
Vesicular Transport ◽  
Super Resolution ◽  
Glycosyl Transferases ◽  
Golgi Transport ◽  
Transport Vesicle ◽  
Membrane Bound ◽  
Soluble Aggregates ◽  
Super Resolution Microscopy

A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary.

scholarly journals Dissecting the Connexin43 Vesicular Transport Pathway by Super-Resolution Microscopy

2016 ◽  
Vol 22 (S3) ◽  
pp. 1012-1013
Author(s):  
Michael J. Zeitz ◽  
Carissa C. James ◽  
James W. Smyth
Keyword(s):  
Vesicular Transport ◽  
Super Resolution ◽  
Transport Pathway ◽  
Super Resolution Microscopy

scholarly journals Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent inflammatory responses

2021 ◽  
Author(s):  
David Klenerman ◽  
Jonathan Meng ◽  
Yu Zhang ◽  
Dominik Saman ◽  
Suman De ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Mechanistic Model ◽  
Super Resolution ◽  
Hyperphosphorylated Tau ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Toll Like Receptor 4 ◽  
Soluble Aggregates ◽  
Super Resolution Microscopy

Abstract Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by PKA in conjugation with either GSK3-β or SAPK4 enabled recombinant wild-type (WT) tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites and the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, in comparison with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4 (TLR4)-dependent inflammatory responses. Together, our results demonstrate that tau hyperphosphorylation is potentially damaging to cells, providing a mechanistic model of how hyperphosphorylation of tau aggregates drives neuroinflammation in tauopathies.

scholarly journals Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

2020 ◽  
Author(s):  
Tobias C. Kunz ◽  
Ralph Götz ◽  
Shiqiang Gao ◽  
Markus Sauer ◽  
Vera Kozjak-Pavlovic
Keyword(s):  
Morphological Changes ◽  
Membrane Surface ◽  
Cell Signalling ◽  
Super Resolution ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Intermembrane Space ◽  
Membrane Bound ◽  
Super Resolution Microscopy ◽  
First Time

AbstractMitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signalling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to ageing and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED and SIM have been applied for cristae visualisation.Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labelled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labelling and ExM as a tool for studying internal structure of mitochondria.

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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