scholarly journals Amyloid-beta oligomers induce Parkin-mediated mitophagy by reducing Miro1

2020 ◽  
Author(s):  
Min Kyoung Kam ◽  
Dong Gil Lee ◽  
Bokyung Kim ◽  
Jae-Won Huh ◽  
H.J. Lee ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Neuronal Cell ◽  
Time Lapse ◽  
Mitochondrial Fragmentation ◽  
Mitochondrial Quality Control ◽  
Cell Functions ◽  
Mitochondrial Motility ◽  
Ros Scavenger ◽  
Time Lapse Imaging

Alzheimer’s disease (AD) is a neurodegenerative disease associated with the accumulation of amyloid-beta oligomers (AβOs). Recent studies have demonstrated that mitochondria-specific autophagy (mitophagy) contributes to mitochondrial quality control by selectively eliminating the dysfunctional mitochondria. Mitochondria motility, which is regulated by Miro1, is also associated with neuronal cell functions. However, the role played by Miro1 in the mitophagy mechanism, especially relative to AβOs and neurodegenerative disorders, remains unknown. In this study, AβOs induced mitochondrial dysfunction, enhanced Parkin-mediated mitophagy, and reduced mitochondrial quantities in hippocampal neuronal cells (HT-22 cells). We demonstrated that AβO-induced mitochondrial fragmentation could be rescued to the elongated mitochondrial form and that mitophagy could be mitigated by the stable overexpression of Miro1 or by pretreatment with N-acetylcysteine (NAC)-a reactive oxygen species (ROS) scavenger-as assessed by immunocytochemistry. Moreover, using time-lapse imaging, under live cell-conditions, we verified that mitochondrial motility was rescued by the Miro1 overexpression. Finally, in HT-22 cells from amyloid precursor protein (APP)/presenilin 1 (PS1)/Tau triple-transgenic mice, we noted that the co-localization between mitochondria and LC3B puncta increased. Taken together, these results indicated that upregulated ROS, induced by AβO, increased the degree of mitophagy and decreased the Miro1 expression levels. In contrast, the Miro1 overexpression ameliorated AβO-mediated mitophagy and increased the mitochondrial motility. In AD model mice, AβOs induced mitophagy in the hippocampus. Thus, our results would improve our understanding of the role of mitophagy in AD toward facilitating the development of novel therapeutic agents for the treatment of AβO-mediated diseases.

scholarly journals Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chiara Beretta ◽  
Elisabeth Nikitidou ◽  
Linn Streubel-Gallasch ◽  
Martin Ingelsson ◽  
Dag Sehlin ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cortical Neurons ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Time Lapse ◽  
Cellular Mechanisms ◽  
Axonal Swelling ◽  
Tem Analysis ◽  
Neuronal Cell Bodies ◽  
Time Lapse Imaging

AbstractAlzheimer’s disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ42 protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aβ42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from Aβ42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aβ exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathology.

scholarly journals Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth in Streptomyces coelicolor

2008 ◽  
Vol 190 (22) ◽  
pp. 7579-7583 ◽  
Author(s):  
Antje Marie Hempel ◽  
Sheng-bing Wang ◽  
Michal Letek ◽  
José A. Gil ◽  
Klas Flärdh
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Time Lapse ◽  
Cell Wall Assembly ◽  
Peptidoglycan Synthesis ◽  
Wall Growth ◽  
Lateral Branches ◽  
Wall Assembly ◽  
Time Lapse Imaging

ABSTRACT Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.

scholarly journals EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

2017 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Cellular Functions ◽  
Binding Ability ◽  
Spatiotemporal Regulation ◽  
Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

scholarly journals Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function

PLoS Biology ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. e3001444
Author(s):  
Nina L. Kikel-Coury ◽  
Jacob P. Brandt ◽  
Isabel A. Correia ◽  
Michael R. O’Dea ◽  
Dana F. DeSantis ◽  
...  
Keyword(s):  
Heart Rate ◽  
Nervous System ◽  
Cardiac Development ◽  
Time Lapse ◽  
Zebrafish Model ◽  
Single Cell Sequencing ◽  
Parasympathetic System ◽  
And Function ◽  
Time Lapse Imaging

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest–derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

scholarly journals A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1

2021 ◽  
Author(s):  
Funmilayo O Fagbadebo ◽  
Philipp D Kaiser ◽  
Katharina Zittlau ◽  
Natascha Bartlick ◽  
Teresa R Wagner ◽  
...  
Keyword(s):  
Time Lapse ◽  
Model Systems ◽  
Live Cells ◽  
Mitochondrial Outer Membrane ◽  
Potential Biomarker ◽  
Time Lapse Imaging ◽  
Lateral Sclerosis ◽  
Neuronal Disorders ◽  
Research Tools

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

scholarly journals JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration

2021 ◽  
Vol 9 ◽  
Author(s):  
Sruthi Alahari ◽  
Abby Farrell ◽  
Leonardo Ermini ◽  
Chanho Park ◽  
Julien Sallais ◽  
...  
Keyword(s):  
Time Lapse ◽  
Lysosomal Degradation ◽  
Related Disorder ◽  
Novel Target ◽  
And Function ◽  
Time Lapse Imaging ◽  
Fibronectin Assembly ◽  
Degradation Time ◽  
Trophoblast Migration

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

scholarly journals Completion of BAX recruitment correlates with mitochondrial fission during apoptosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
M. E. Maes ◽  
J. A. Grosser ◽  
R. L. Fehrman ◽  
C. L. Schlamp ◽  
R. W. Nickells
Keyword(s):  
Mitochondrial Fission ◽  
Late Phase ◽  
Time Lapse ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Fragmentation ◽  
Fission Process ◽  
Bax Protein ◽  
Functional Part ◽  
Time Lapse Imaging ◽  
Fully Functioning

Abstract BAX, a member of the BCL2 gene family, controls the committed step of the intrinsic apoptotic program. Mitochondrial fragmentation is a commonly observed feature of apoptosis, which occurs through the process of mitochondrial fission. BAX has consistently been associated with mitochondrial fission, yet how BAX participates in the process of mitochondrial fragmentation during apoptosis remains to be tested. Time-lapse imaging of BAX recruitment and mitochondrial fragmentation demonstrates that rapid mitochondrial fragmentation during apoptosis occurs after the complete recruitment of BAX to the mitochondrial outer membrane (MOM). The requirement of a fully functioning BAX protein for the fission process was demonstrated further in BAX/BAK-deficient HCT116 cells expressing a P168A mutant of BAX. The mutant performed fusion to restore the mitochondrial network. but was not demonstrably recruited to the MOM after apoptosis induction. Under these conditions, mitochondrial fragmentation was blocked. Additionally, we show that loss of the fission protein, dynamin-like protein 1 (DRP1), does not temporally affect the initiation time or rate of BAX recruitment, but does reduce the final level of BAX recruited to the MOM during the late phase of BAX recruitment. These correlative observations suggest a model where late-stage BAX oligomers play a functional part of the mitochondrial fragmentation machinery in apoptotic cells.

scholarly journals Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth.

1994 ◽  
Vol 127 (4) ◽  
pp. 1071-1084 ◽  
Author(s):  
B Varnum-Finney ◽  
L F Reichardt
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Cell Lines ◽  
Growth Cone ◽  
Pc12 Cell ◽  
Neuronal Cell ◽  
Time Lapse ◽  
Proximal Region ◽  
Normal Numbers

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin-deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.

scholarly journals Role of turgor pressure in endocytosis in fission yeast

2014 ◽  
Vol 25 (5) ◽  
pp. 679-687 ◽  
Author(s):  
Roshni Basu ◽  
Emilia Laura Munteanu ◽  
Fred Chang
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Fission Yeast ◽  
Turgor Pressure ◽  
Time Lapse ◽  
Actin Assembly ◽  
Latrunculin A ◽  
The Media ◽  
Time Lapse Imaging

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.

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