scholarly journals The heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of mitochondrial dynamics and ER contact sites, regulate heme mobilization to the nucleus

2019 ◽  
Author(s):  
Osiris Martinez-Guzman ◽  
Mathilda M. Willoughby ◽  
Arushi Saini ◽  
Jonathan V. Dietz ◽  
Iryna Bohovych ◽  
...  
Keyword(s):  
Distribution Systems ◽  
Temporal Dynamics ◽  
Aminolevulinic Acid ◽  
Mitochondrial Dynamics ◽  
Live Cell ◽  
Mitochondrial Matrix ◽  
Biosynthetic Enzyme ◽  
Signaling Molecule ◽  
Contact Sites ◽  
Cell Assay

AbstractHeme is an iron-containing cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. We found that heme distribution occurs simultaneously via parallel pathways. In fact, surprisingly, we find that trafficking to the nucleus is ∼25% faster than to the cytosol or mitochondrial matrix. Moreover, we discovered that the heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Altogether, our results indicate that the nucleus acquires heme faster than the cytosol or mitochondrial matrix, presumably for mitochondrial-nuclear retrograde signaling, and that GTPases that regulate mitochondrial dynamics and ER contact sites are hard-wired to cellular heme distribution systems.Summary StatementThe factors that govern the trafficking of heme, an essential but potentially cytotoxic cofactor and signaling molecule, are poorly understood. Herein, we developed a live-cell assay to monitor heme distribution kinetics and identified the first enzyme in the heme synthesis pathway and GTPases in control of mitochondrial-ER contact sites and dynamics as being critical modulators of heme trafficking.

scholarly journals Mitochondrial-nuclear heme trafficking is regulated by GTPases that control mitochondrial dynamics

2019 ◽  
Author(s):  
Osiris Martinez-Guzman ◽  
Jonathan V. Dietz ◽  
Iryna Bohovych ◽  
Amy E. Medlock ◽  
Oleh Khalimonchuk ◽  
...  
Keyword(s):  
Temporal Dynamics ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Matrix ◽  
Mitochondrial Network ◽  
Negative Regulators ◽  
Subcellular Compartment ◽  
The Matrix ◽  
Spatio Temporal ◽  
Heme Trafficking ◽  
Dependent Processes

AbstractHeme is an essential cofactor and signaling molecule. All heme-dependent processes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins in virtually every subcellular compartment. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. To address this, we developed a pulse-chase assay in which, upon the initiation of heme synthesis, heme mobilization into the mitochondrial matrix, cytosol and nucleus is monitored using fluorescent heme sensors. Surprisingly, we found that heme trafficking to the nucleus occurs at a faster rate than to the matrix or cytosol. Further, we demonstrate that GTPases in control of mitochondrial fusion, Mgm1, and fission, Dnm1, are positive and negative regulators of mitochondrial-nuclear heme trafficking, respectively. We also find that heme controls mitochondrial network morphology. Altogether, our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.

A matrix targeted fluorescent probe to monitor mitochondrial dynamics

2021 ◽  
Author(s):  
Madhu Ramesh ◽  
Kolla Rajasekhar ◽  
Kavya Gupta ◽  
Vardhaman Babagond ◽  
Deepak Kumar Saini ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Chemical Stability ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Matrix ◽  
Turn On

A far-red turn-on fluorescent probe (Mito-TG) with excellent biocompatibility, photostability, chemical stability targets mitochondrial matrix. The insensitivity of probe under different pH and ROS enabled tracking of mitophagy and Aβ induced mitochondrial dynamics.

scholarly journals hFUT1-Based Live-Cell Assay To Profile α1-2-Fucoside-Enhanced Influenza Virus A Infection

2020 ◽  
Vol 15 (4) ◽  
pp. 819-823 ◽  
Author(s):  
Senlian Hong ◽  
Geramie Grande ◽  
Chenhua Yu ◽  
Digantkumar G. Chapla ◽  
Natalie Reigh ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Live Cell ◽  
Influenza Virus A ◽  
Cell Assay

scholarly journals Miga-mediated endoplasmic reticulum–mitochondria contact sites regulate neuronal homeostasis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lingna Xu ◽  
Xi Wang ◽  
Jia Zhou ◽  
Yunyi Qiu ◽  
Weina Shang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Mitochondrial Dynamics ◽  
Molecular Organization ◽  
Lipid Transport ◽  
Cellular Processes ◽  
Contact Sites ◽  
Neuronal Homeostasis ◽  
Higher Eukaryotes ◽  
Lateral Sclerosis

Endoplasmic reticulum (ER)–mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.

scholarly journals Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia R Kardon ◽  
Jamie A Moroco ◽  
John R Engen ◽  
Tania A Baker
Keyword(s):  
Active Site ◽  
Autonomous System ◽  
Aminolevulinic Acid ◽  
Structural Features ◽  
Biosynthetic Enzyme ◽  
Cofactor Binding ◽  
Partial Unfolding ◽  
Chaperone Interactions ◽  
Aaa Protein ◽  
Insight Into

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

scholarly journals DRP1 regulates endoplasmic reticulum sheets to control mitochondrial DNA replication and segregation

2021 ◽  
Author(s):  
Hema Saranya Ilamathi ◽  
Sara Benhammouda ◽  
Justine Desrochers-Goyette ◽  
Matthew A Lines ◽  
Marc Germain
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitochondrial Dna ◽  
Protein Complexes ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Diseases ◽  
Contact Sites ◽  
Transport Chain ◽  
Essential Components ◽  
Mtdna Replication

Mitochondria are multi-faceted organelles crucial for cellular homeostasis that contain their own genome. Mitochondrial DNA (mtDNA) codes for several essential components of the electron transport chain, and mtDNA maintenance defects lead to mitochondrial diseases. mtDNA replication occurs at endoplasmic reticulum (ER)-mitochondria contact sites and is regulated by mitochondrial dynamics. Specifically, mitochondrial fusion is essential for mtDNA maintenance. In contrast, while loss of mitochondrial fission causes the aggregation of nucleoids (mtDNA-protein complexes), its role in nucleoid distribution remains unclear. Here, we show that the mitochondrial fission protein DRP1 regulates nucleoid segregation by altering ER sheets, the ER structure associated with protein synthesis. Specifically, DRP1 loss or mutation leads to altered ER sheets that physically interact with mitobulbs, mitochondrial structures containing aggregated nucleoids. Importantly, nucleoid distribution and mtDNA replication were rescued by expressing the ER sheet protein CLIMP63. Thus, our work identifies a novel mechanism by which DRP1 regulates mtDNA replication and distribution.

scholarly journals A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Yuki Takamatsu ◽  
Olga Dolnik ◽  
Takeshi Noda ◽  
Stephan Becker
Keyword(s):  
Live Cell Imaging ◽  
Intracellular Transport ◽  
Actin Polymerization ◽  
Ebola Virus ◽  
Cell Imaging ◽  
Temporal Dynamics ◽  
Imaging System ◽  
Marburg Virus ◽  
Live Cell ◽  
Infected Cells

Abstract Background Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. Methods To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Results Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. Conclusions We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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