Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

Disturbed Endothelial Blood-Bone Marrow-Barrier In Nox4 Deficient Mice

Reactive oxygen species (ROS) have been implicated in the regulation of stemness of hematopoietic stem cells (HSC). HSC with long-term repopulating capabilities are characterized by low ROS levels, whereas increased ROS levels correlate with lineage specification and differentiation. Several tightly regulated sources of ROS production are well known among which are the NADPH oxidases (Nox). HSC are known to express Nox1, Nox2 and Nox4, however, their role in maintenance of stem cell potential or in the activation of differentiation programs are poorly understood. While Nox2 is activated in response to various extrinsic and intrinsic stimuli, mainly during infection and inflammation, Nox4 is constitutively active and is considered to be responsible for steady-state ROS production. Consequently, Nox4 deficiency might lower ROS levels at steady-state hematopoiesis and thereby could have an impact on HSC physiology. In this work we studied HSC homeostasis in Nox4 knock-out mice. Analysis of the hematopoietic stem and progenitor cell (HSPC) pool in the bone marrow (BM) revealed no significant differences in the levels of Lineage marker negative (Lin-) Sca-1+ ckit+ (LSK) and LSK-SLAM (LSK CD150+ CD48-) cells in Nox4 deficient mice compared to wild type (WT) C57BL/6J mice. HSPC frequency upon primary and secondary BM transplantation was comparable between Nox4 deficient and WT mice. In addition, the frequency of colony forming cells in the BM under steady-state conditions did not differ between both mouse groups. However, Nox4 deficient mice possess more functional HSCs as observed in in vivo competitive repopulating unit (CRU) assays. Lin- cells derived from Nox4 knock out (KO) mice showed an increased CRU frequency and superior multilineage engraftment upon secondary transplantation. Surprisingly, ROS levels in different HSPC subsets of NOX4 KO mice were comparable to WT cells, implying that the absence of Nox4 in HSCs does not have a major intrinsic impact on HSC physiology via ROS. Therefore, the increased levels of functional HSCs observed in our studies may suggest a contribution of the BM microenvironment to steady-state hematopoiesis in the BM of Nox4 KO animals. Recent observations suggest a regulation of the BM stem cell pool by BM endothelial cells, in particular by the permeability state of the blood-bone marrow-barrier (Itkin T et al., ASH Annual Meeting Abstracts, 2012). Endothelial cells interact with HSCs predominantly via paracrine effects and control stem cell retention, egress and homing as well as stem cell activation. As Nox4 is highly expressed in endothelial cells and is involved in angiogenesis, we reasoned that the absence of NOX4 could affect HSC homeostasis through altered BM endothelium properties and barrier permeability state. Indeed, in preliminary assays we found reduced short-term homing of BM mononuclear cells into the BM of Nox4 deficient mice as compared to wild type hosts. Furthermore, in vivo administration of Evans Blue dye revealed reduced dye penetration into Nox4-/- BM compared to wild type mice upon intravenous injection. Taken together, these data indicate a reduced endothelial permeability in Nox4 KO mice. Ongoing experiments aim at further characterization of the Nox4-/- phenotype in BM sinusoidal and arteriolar endothelial cells, the impact of Nox4 deletion on BM hematopoietic and mesenchymal stem cells, and in deciphering the role of Nox4 in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7channel

Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3′- O-(4-benzoyl)benzoyl-adenosine 5′-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5′-triphosphate, or adenosine 5′- O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 μM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 μM) at 37°C, and the delay was longer at 24°C. ATP addition 2–10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10–20 μM). P2Z/P2X7 inhibitors, KN-62 and 5-( N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.

ATP depletion inhibits capacitative Ca2+ entry in rat thymic lymphocytes

The present study investigates the requirement for cellular ATP in the increase in plasma membrane Ca2+ permeability activated by the release of Ca2+ from intracellular stores in rat thymic lymphocytes (capacitative Ca2+ entry). The permeability state of this pathway following activation with thapsigargin was probed in control and ATP-depleted cells using fluorometric measurements of intracellular Ca2+, Mn2+ entry, and membrane potential, and unidirectional measurements of Ca2+ uptake using 45Ca2+. The capacitative Ca(2+)-entry pathway was markedly inhibited in cells depleted of ATP by incubation in glucose-free solution containing oligomycin, antimycin A, and 2-deoxy-D-glucose. These data cannot be explained on the basis of a loss of the transmembrane electrochemical gradient for Ca2+, alterations in intracellular pH or cellular Na+ content, a direct effect of the inhibitors of ATP production on the capacitative Ca(2+)-entry pathway, or the ability of thapsigargin to release Ca2+ from intracellular stores. Rather, these data are consistent with a requirement for ATP or a high-energy phosphate donor in the activation and/or maintained activation of capacitative Ca2+ entry.

Palestine and the Arab State System: Permeability, State Consolidation and the Intifada

This article examines the sensitivity of Arab states to the political and ideological repercussions of the Palestine issue by focussing upon Egypt, Syria and Jordan. It suggests that the policies of Arab regimes towards the Palestine issue have been substantially shaped by historical patterns of state formation, and by the gradual consolidation of the Arab state system. This has served to “harden” the Arab territorial state, creating conditions under which Arab states are increasingly (if only partially) insulated from the transnational effects of the Palestinian-Israeli conflict.