PMRT1, a Plasmodium specific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and showed essentiality of four out of the six membrane transporters during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis. Heterologous expression of wild-type and mutation constructs in Xenopus laevis oocytes indicated ion transport upon membrane hyperpolarization and a functional role of negatively charged amino acids protruding into the parasitophorous vacuole lumen. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development and provide the first functional characterization of PfPMRT1, an essential parasite membrane transporter.

Fungal Mineral Weathering Mechanisms Revealed Through Direct Molecular Visualization

Soil fungi facilitate the translocation of inorganic nutrients from soil minerals to other microorganisms and plants. This ability is particularly advantageous in impoverished soils, because fungal mycelial networks can bridge otherwise spatially disconnected and inaccessible nutrient hotspots. However, the molecular mechanisms underlying fungal mineral weathering and transport through soil remains poorly understood. Here, we addressed this knowledge gap by directly visualizing nutrient acquisition and transport through fungal hyphae in a mineral doped soil micromodel using a multimodal imaging approach. We observed that Fusarium sp. DS 682, a representative of common saprotrophic soil fungi, exhibited a mechanosensory response (thigmotropism) around obstacles and through pore spaces (~12 μm) in the presence of minerals. The fungus incorporated and translocated potassium (K) from K-rich mineral interfaces, as evidenced by visualization of mineral derived nutrient transport and unique K chemical moieties following fungal induced mineral weathering. Specific membrane transport proteins were expressed in the presence of minerals, including those involved in oxidative phosphorylation pathways and transmembrane transport of small molecular weight organic acids. This study establishes the significance of fungal biology and nutrient translocation mechanisms in maintaining fungal growth under water and nutrient limitations in a soil-like microenvironment.

Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Heat Stress-Dependent Association of Membrane Trafficking Proteins With mRNPs Is Selective

The Arabidopsis AAA ATPase SKD1 is essential for ESCRT-dependent endosomal sorting by mediating the disassembly of the ESCRTIII complex in an ATP-dependent manner. In this study, we show that SKD1 localizes to messenger ribonucleoprotein complexes upon heat stress. Consistent with this, the interactome of SKD1 revealed differential interactions under normal and stress conditions and included membrane transport proteins as well as proteins associated with RNA metabolism. Localization studies with selected interactome proteins revealed that not only RNA associated proteins but also several ESCRTIII and membrane trafficking proteins were recruited to messenger ribonucleoprotein granules after heat stress.

Extended Bloch–McConnell equations for mechanistic analysis of hyperpolarized ¹³C magnetic resonance experiments on enzyme systems

We describe an approach to formulating the kinetic master equations of the time evolution of NMR signals in reacting (bio)chemical systems. Special focus is given to studies that employ signal enhancement (hyperpolarization) methods such as dissolution dynamic nuclear polarization (dDNP) and involving nuclear spin-bearing solutes that undergo reactions mediated by enzymes and membrane transport proteins. We extend the work given in a recent presentation on this topic (Kuchel and Shishmarev, 2020) to now include enzymes with two or more substrates and various enzyme reaction mechanisms as classified by Cleland, with particular reference to non-first-order processes. Using this approach, we can address some pressing questions in the field from a theoretical standpoint. For example, why does binding of a hyperpolarized substrate to an enzyme not cause an appreciable loss of the signal from the substrate or product? Why does the concentration of an unlabelled pool of substrate, for example 12C lactate, cause an increase in the rate of exchange of the 13C-labelled pool? To what extent is the equilibrium position of the reaction perturbed during administration of the substrate? The formalism gives a full mechanistic understanding of the time courses derived and is of relevance to ongoing clinical trials using these techniques.

Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.