scholarly journals Remodeling of ER-exit sites initiates a membrane supply pathway for autophagosome biogenesis

2017 ◽  
Author(s):  
Liang Ge ◽  
Min Zhang ◽  
Samuel J Kenny ◽  
Dawei Liu ◽  
Miharu Maeda ◽  
...  
Keyword(s):  
Super Resolution ◽  
Membrane Vesicles ◽  
Membrane Component ◽  
Autophagosome Formation ◽  
Er Exit Sites ◽  
Intracellular Compartments ◽  
A Subunit ◽  
Intermediate Compartment ◽  
Copii Vesicles ◽  
Super Resolution Microscopy

AbstractAutophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.

scholarly journals FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

2020 ◽  
Author(s):  
Eleonora Turco ◽  
Irmgard Fischer ◽  
Sascha Martens
Keyword(s):  
Kinase Activity ◽  
De Novo ◽  
Super Resolution ◽  
Membrane Vesicles ◽  
Degradation Pathway ◽  
Autophagosome Formation ◽  
Ubiquitinated Proteins ◽  
The Many ◽  
Super Resolution Microscopy ◽  
Insight Into

AbstractMacroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.

scholarly journals Super-resolution microscopy unveils FIP200-scaffolded, cup-shaped organization of mammalian autophagic initiation machinery

2019 ◽  
Author(s):  
Samuel J Kenny ◽  
Xuyan (Shirley) Chen ◽  
Liang Ge ◽  
Ke Xu
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Subcellular Location ◽  
Outer Face ◽  
Structural Protein ◽  
Er Exit Sites ◽  
Autophagy Pathway ◽  
20 Nm ◽  
Yeast Homolog ◽  
Super Resolution Microscopy

AbstractAutophagy is an essential physiological process by which eukaryotic cells degrade and recycle cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway have been identified, questions remain regarding the sequence, subcellular location, and structural requirements of autophagosome formation. Here, we characterize the structural organization of key components of the mammalian autophagic initiation machinery at ∼20 nm spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a structural asymmetry to enforce the directional recruitment of downstream components, including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we provide evidence that the early autophagic machinery is recruited in its entirety to these cup structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the phagophore membrane during elongation. We thus shed new light on the physical process of mammalian autophagic initiation and development at the nanometer-scale.

scholarly journals Subcellular drug targeting illuminates local kinase action

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Paula J Bucko ◽  
Chloe K Lombard ◽  
Lindsay Rathbun ◽  
Irvin Garcia ◽  
Akansha Bhat ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Super Resolution ◽  
Biological Processes ◽  
Anchoring Proteins ◽  
Intracellular Compartments ◽  
Mitotic Spindle Assembly ◽  
Targeted Inhibition ◽  
Super Resolution Microscopy

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

scholarly journals COPII vesicles contribute to autophagosomal membranes

2019 ◽  
Vol 218 (5) ◽  
pp. 1503-1510 ◽  
Author(s):  
Takayuki Shima ◽  
Hiromi Kirisako ◽  
Hitoshi Nakatogawa
Keyword(s):  
De Novo ◽  
Membrane Vesicles ◽  
Membrane Dynamics ◽  
Fundamental Question ◽  
Autophagosome Formation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Definitive Answer ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Labeling System

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle–labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

scholarly journals New insights into protein secretion: TANGO1 runs rings around the COPII coat

2017 ◽  
Vol 216 (4) ◽  
pp. 859-861 ◽  
Author(s):  
Benjamin S. Glick
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Secretion ◽  
Super Resolution ◽  
Cell Biol ◽  
Er Exit Sites ◽  
Er Export ◽  
Super Resolution Microscopy

In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611088) and Raote et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608080) use super-resolution microscopy to visualize large COPII-coated endoplasmic reticulum (ER) export carriers. Rings of TANGO1 surround COPII, implicating TANGO1 in organizing ER exit sites and in regulating COPII coat dynamics and geometry.

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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