Analysis of Endoplasmic Reticulum–Endosome Association Using Live-Cell Imaging in Plant Cells

2020 ◽  
pp. 23-33
Author(s):  
Giovanni Stefano ◽  
Federica Brandizzi
Keyword(s):  
Endoplasmic Reticulum ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Plant Cells ◽  
Live Cell

scholarly journals EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

2018 ◽  
Vol 217 (6) ◽  
pp. 2047-2058 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Carlo Giovanni Quintanilla ◽  
Ting-Sung Hsieh ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Functions ◽  
Binding Ability ◽  
Trapping Mechanism ◽  
Spatiotemporal Regulation

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER–plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1–EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER–PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER–PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1–EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

scholarly journals Live Cell Imaging of Protein Dislocation from the Endoplasmic Reticulum

2012 ◽  
Vol 287 (33) ◽  
pp. 28057-28066 ◽  
Author(s):  
Yongwang Zhong ◽  
Shengyun Fang
Keyword(s):  
Endoplasmic Reticulum ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

scholarly journals Impaired mitochondrial–endoplasmic reticulum interaction and mitophagy in Miro1-mutant neurons in Parkinson’s disease

2020 ◽  
Vol 29 (8) ◽  
pp. 1353-1364 ◽  
Author(s):  
Clara Berenguer-Escuder ◽  
Dajana Grossmann ◽  
Paul Antony ◽  
Giuseppe Arena ◽  
Kobi Wasner ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Endoplasmic Reticulum ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Mitochondrial Dynamics ◽  
Live Cell ◽  
Calcium Handling ◽  
Imaging Results ◽  
And Function

Abstract Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson’s disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

scholarly journals The Fusome Mediates Intercellular Endoplasmic Reticulum Connectivity in Drosophila Ovarian Cysts

2004 ◽  
Vol 15 (10) ◽  
pp. 4512-4521 ◽  
Author(s):  
Erik L. Snapp ◽  
Takako Iida ◽  
David Frescas ◽  
Jennifer Lippincott-Schwartz ◽  
Mary A. Lilly
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Ovarian Cysts ◽  
Cleavage Furrow ◽  
Cytoskeletal Component ◽  
The Common ◽  
Oocyte Differentiation

Drosophila ovarian cysts arise through a series of four synchronous incomplete mitotic divisions. After each round of mitosis, a membranous organelle, the fusome, grows along the cleavage furrow and the remnants of the mitotic spindle to connect all cystocytes in a cyst. The fusome is essential for the pattern and synchrony of the mitotic cyst divisions as well as oocyte differentiation. Using live cell imaging, greenfluorescent protein–tagged proteins, and photobleaching techniques, we demonstrate that fusomal endomembranes are part of a single continuous endoplasmic reticulum (ER) that is shared by all cystocytes in dividing ovarian cysts. Membrane and lumenal proteins of the common ER freely and rapidly diffuse between cystocytes. The fusomal ER mediates intercellular ER connectivity by linking the cytoplasmic ER membranes of all cystocytes within a cyst. Before entry into meiosis and onset of oocyte differentiation (between region 1 and region 2A), ER continuity between cystocytes is lost. Furthermore, analyses of hts and Dhc64c mutants indicate that intercellular ER continuity within dividing ovarian cysts requires the fusome cytoskeletal component and suggest a possible role for the common ER in synchronizing mitotic cyst divisions.

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