scholarly journals The use of the Ca2+-sensitive intramitochondrial dehydrogenases and entrapped fura-2 to study Sr2+ and Ba2+ transport across the inner membrane of mammalian mitochondria

1990 ◽  
Vol 192 (1) ◽  
pp. 239-244 ◽  
Author(s):  
James G. McCORMACK ◽  
Nicola J. OSBALDESTON
Keyword(s):  
Inner Membrane ◽  
Fura 2 ◽  
Mammalian Mitochondria

scholarly journals Cytosolic Ni(II) Sensor in Cyanobacterium

2012 ◽  
Vol 287 (15) ◽  
pp. 12142-12151 ◽  
Author(s):  
Andrew W. Foster ◽  
Carl J. Patterson ◽  
Rafael Pernil ◽  
Corinna R. Hess ◽  
Nigel J. Robinson
Keyword(s):  
Coordination Sphere ◽  
Promoter Region ◽  
Nitrilotriacetic Acid ◽  
The Other ◽  
Cryptic Promoter ◽  
Difference Spectra ◽  
Inner Membrane ◽  
Fura 2 ◽  
Metal Selectivity ◽  
Metal Sensors

Efflux of surplus Ni(II) across the outer and inner membranes of Synechocystis PCC 6803 is mediated by the Nrs system under the control of a sensor of periplasmic Ni(II), NrsS. Here, we show that the product of ORF sll0176, which encodes a CsoR/RcnR-like protein now designated InrS (for internal nickel-responsive sensor), represses nrsD (NrsD is deduced to efflux Ni(II) across the inner membrane) from a cryptic promoter between the final two ORFs in the nrs operon. Transcripts initiated from the newly identified nrsD promoter accumulate in response to nickel or cobalt but not copper, and recombinant InrS forms specific, Ni(II)-inhibited complexes with the nrsD promoter region. Metal-dependent difference spectra of Ni(II)- and Cu(I)-InrS are similar to Cu(I)-sensing CsoR and dissimilar to Ni(II)/Co(II)-sensing RcnR, consistent with factors beyond the primary coordination sphere switching metal selectivity. Competition with chelators mag-fura-2, nitrilotriacetic acid, EDTA, and EGTA estimate KD Ni(II) for the tightest site of InrS as 2.05 (±1.5) × 10−14m, and weaker KD Ni(II) for the cells' metal sensors of other types: Zn(II) co-repressor Zur, Co(II) activator CoaR, and Zn(II) derepressor ZiaR. Ni(II) transfer to InrS occurs upon addition to Ni(II) forms of each other sensor. InrS binds Ni(II) sufficiently tightly to derepress Ni(II) export at concentrations below KD Ni(II) of the other sensors.

scholarly journals Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

2009 ◽  
Vol 20 (19) ◽  
pp. 4216-4224 ◽  
Author(s):  
Mirko Koppen ◽  
Florian Bonn ◽  
Sarah Ehses ◽  
Thomas Langer
Keyword(s):  
Neurodegenerative Disorders ◽  
Intermediate Form ◽  
Inner Membrane ◽  
Processing Enzymes ◽  
Mitochondrial Processing Peptidase ◽  
Mammalian Mitochondria ◽  
Autocatalytic Processing ◽  
Processing Peptidase ◽  
Neuronal Disorders

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.

scholarly journals Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion

2009 ◽  
Vol 20 (15) ◽  
pp. 3525-3532 ◽  
Author(s):  
Zhiyin Song ◽  
Mariam Ghochani ◽  
J. Michael McCaffery ◽  
Terrence G. Frey ◽  
David C. Chan
Keyword(s):  
Membrane Fusion ◽  
Outer Membrane ◽  
Null Mouse ◽  
Tight Coupling ◽  
Inner Membrane ◽  
Mammalian Mitochondria ◽  
Mouse Cells ◽  
Large Gtpases

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases—OPA1 and the mitofusins Mfn1 and Mfn2—are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.

scholarly journals Mic60 exhibits a coordinated clustered distribution along and across yeast and mammalian mitochondria

2019 ◽  
Vol 116 (20) ◽  
pp. 9853-9858 ◽  
Author(s):  
Stefan Stoldt ◽  
Till Stephan ◽  
Daniel C. Jans ◽  
Christian Brüser ◽  
Felix Lange ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Interaction Network ◽  
Yeast Mitochondria ◽  
Contact Site ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Mammalian Mitochondria ◽  
Interaction Partners ◽  
A Subunit

Mitochondria are tubular double-membrane organelles essential for eukaryotic life. They form extended networks and exhibit an intricate inner membrane architecture. The MICOS (mitochondrial contact site and cristae organizing system) complex, crucial for proper architecture of the mitochondrial inner membrane, is localized primarily at crista junctions. Harnessing superresolution fluorescence microscopy, we demonstrate that Mic60, a subunit of the MICOS complex, as well as several of its interaction partners are arranged into intricate patterns in human and yeast mitochondria, suggesting an ordered distribution of the crista junctions. We show that Mic60 forms clusters that are preferentially localized in the inner membrane at two opposing sides of the mitochondrial tubules so that they form extended opposing distribution bands. These Mic60 distribution bands can be twisted, resulting in a helical arrangement. Focused ion beam milling-scanning electron microscopy showed that in yeast the twisting of the opposing distribution bands is echoed by the folding of the inner membrane. We show that establishment of the Mic60 distribution bands is largely independent of the cristae morphology. We suggest that Mic60 is part of an extended multiprotein interaction network that scaffolds mitochondria.

scholarly journals A maxi-chloride channel in the inner membrane of mammalian mitochondria

2008 ◽  
Vol 1777 (11) ◽  
pp. 1438-1448 ◽  
Author(s):  
Umberto De Marchi ◽  
Ildikò Szabò ◽  
Grazia M. Cereghetti ◽  
Pranvera Hoxha ◽  
William J. Craigen ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Inner Membrane ◽  
Mammalian Mitochondria

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

1987 ◽  
Vol 58 (02) ◽  
pp. 737-743 ◽  
Author(s):  
Frarnçois Lanza ◽  
Alain Beretz ◽  
Martial Kubina ◽  
Jean-Pierre Cazenave
Keyword(s):  
Intracellular Calcium ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Free Calcium ◽  
Membrane Bilayer ◽  
Fluorescent Indicators ◽  
Fura 2 ◽  
Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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