Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane

Abstract Background Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA – cpnF. Results Independent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P. Conclusions Our studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.https://deepblue.lib.umich.edu/bitstream/2027.42/145158/1/12860_2018_Article_160.pd

Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane

Abstract Background Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA – cpnF. Results Independent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P. Conclusions Our studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.https://deepblue.lib.umich.edu/bitstream/2027.42/145158/1/12860_2018_Article_160.pd

Dual role of Miro protein clusters in mitochondrial cristae organisation and ER-Mitochondria Contact Sites

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts (MEF) we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and endoplasmic reticulum-mitochondria contacts sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality

Data from: Behavioural responses of naked mole rats to acute hypoxia and anoxia

Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals. Other species respond to hypoxia by either escaping the hypoxic environment or drastically decreasing behavioural activity and body temperature (Tb) to conserve energy. However, NMRs rarely leave their underground burrows, which are putatively hypoxic and thermally stable near the NMRs' preferred Tb. Therefore, we asked whether NMRs are able to employ behavioural and thermoregulatory strategies in response to hypoxia despite their need to remain active and the minimal thermal scope in their burrows. We exposed NMRs to progressively deeper levels of hypoxia (from 21 to 0% O2) while measuring their behaviour and Tb. Behavioural activity decreased 40–60% in hypoxia and Tb decreased slightly in moderate hypoxia (5–9%) and then further with deeper hypoxia (3% O2). However, even at 3% O2 NMRs remained somewhat active and warm, and continued to explore their environment. Remarkably, NMRs were active for greater than 90 s in acute anoxia and Tb and metabolic rate decreased rapidly. We conclude that NMRs are adapted to remain awake and functional even at the extremes of their hypoxia-tolerance. This adaptation likely reflects variable and challenging levels of environmental hypoxia in the natural habitat of this species

Materials and Methods from Behavioural responses of naked mole rats to acute hypoxia and anoxia

Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals. Other species respond to hypoxia by either escaping the hypoxic environment or drastically decreasing behavioural activity and body temperature (<i>T</i>_b) to conserve energy. However, NMRs rarely leave their underground burrows, which are putatively hypoxic and thermally stable near the NMRs' preferred <i>T</i>_b. Therefore, we asked whether NMRs are able to employ behavioural and thermoregulatory strategies in response to hypoxia despite their need to remain active and the minimal thermal scope in their burrows. We exposed NMRs to progressively deeper levels of hypoxia (from 21 to 0% O₂) while measuring their behaviour and <i>T</i>_b. Behavioural activity decreased 40–60% in hypoxia and <i>T</i>_b decreased slightly in moderate hypoxia (5–9%) and then further with deeper hypoxia (3% O₂). However, even at 3% O₂ NMRs remained somewhat active and warm, and continued to explore their environment. Remarkably, NMRs were active for greater than 90 s in acute anoxia and <i>T</i>_b and metabolic rate decreased rapidly. We conclude that NMRs are adapted to remain awake and functional even at the extremes of their hypoxia-tolerance. This adaptation likely reflects variable and challenging levels of environmental hypoxia in the natural habitat of this species

Infiltration of ambient PM 2.5 and levels of indoor generated non-ETS PM 2.5 in residences of four European cities

Ambient fine particle (PM 2.5) concentrations are associated with premature mortality and other health effects. Urban populations spend a majority of their time in indoor environments, and thus exposures are modified by building envelopes. Ambient particles have been found to penetrate indoors very efficiently (penetration efficiency P≈1.0), where they are slowly removed by deposition, adsorption, and other mechanisms. Other particles are generated indoors, even in buildings with no obvious sources like combustion devices, cooking, use of aerosol products, etc.. The health effects of indoor generated particles are currently not well understood, and require information on concentrations and exposure levels. The current work apportions residential PM 2.5 concentrations measured in the EXPOLIS study to ambient and non-ambient fractions. The results show that the mean infiltration efficiency of PM 2.5 particles is similar in all four cities included in the analysis, ranging from 0.59 in Helsinki to 0.70 in Athens, with Basle and Prague in between. Mean residential indoor concentrations of ambient particles range from 7 (Helsinki) to 21 μg m -3 (Athens). Based on PM 2.5 decay rates estimated in the US, estimates of air exchange rates and indoor source strengths were calculated. The mean air exchange rate was highest in Athens and lowest in Prague. Indoor source strengths were similar in Athens, Basle and Prague, but lower in Helsinki. Some suggestions of possible determinants of indoor generated non-ETS PM 2.5 were acquired using regression analysis. Building materials and other building and family characteristics were associated with the indoor generated particle levels. A significant fraction of the indoor concentrations remained unexplained. © 2004 Elsevier Ltd. All rights reserved

The Green Tea Catechin Epigallocatechin Gallate (EGCG) Blocks Cell Motility, Chemotaxis and Development in <em>Dictyostelium discoideum</em>

<div><p>Catechins, flavanols found at high levels in green tea, have received significant attention due to their potential health benefits related to cancer, autoimmunity and metabolic disease, but little is known about the mechanisms by which these compounds affect cellular behavior. Here, we assess whether the model organism <i>Dictyostelium discoideum</i> is a useful tool with which to characterize the effects of catechins. Epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, has significant effects on the <i>Dictyostelium</i> life cycle. In the presence of EGCG aggregation is delayed, cells do not stream and development is typically stalled at the loose aggregate stage. The developmental effects very likely result from defects in motility, as EGCG reduces both random movement and chemotaxis of <i>Dictyostelium</i> amoebae. These results suggest that catechins and their derivatives may be useful tools with which to better understand cell motility and development in <i>Dictyostelium</i> and that this organism is a useful model to further characterize the activities of catechins.</p> </div