Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria

Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism Podospora anserina revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.     

Macromolecular organization of ATP synthase and complex I in whole mitochondria

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.     

In-cell structures of conserved supramolecular protein arrays at the mitochondria–cytoskeleton interface in mammalian sperm

Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

In many cell types, mitochondria collectively form a dynamic network whose members divide, fuse, and communicate with one another (1–3). Through interactions with the cytoskeleton, mitochondria are transported—sometimes across large distances—and positioned in response to dynamic stimuli (4, 5). Interactions with the cytoskeleton can also restrain mitochondria to specific subcellular locations. In neurons, axonal mitochondria can be immobilized by interactions with the microtubule or actin cytoskeletons (6–8). In cardiac and skeletal muscle, mitochondrial distribution is regulated by interactions with myofibrils and intermediate filaments (9, 10). However, despite the prevalence of intermitochondria and mitochondria–cytoskeleton interactions and their integral roles in cellular function, there is very little information on the molecular architectures of these interaction sites in any cell type.One of the most striking mitochondrial configurations occurs in amniote sperm, in which mitochondria are arranged in a spiral around the axoneme, defining a region called the midpiece (11, 12). Mitochondria are among the few organelles retained in sperm throughout their maturation, during which they otherwise lose most of their cytoplasm and organelles en route to becoming highly streamlined cells specialized for finding and fusing with the egg. The extensive mitochondrial sheath in amniote sperm may be an adaptation needed to power the large, long flagellum in these lineages. Variations in midpiece morphometry affect sperm motility and competitiveness (13, 14), and different species rely on energy from mitochondrial respiration to different extents (15, 16), warranting comparative studies of mitochondrial structure across species.The core of the midpiece is the flagellar cytoskeleton, composed of the microtubule-based axoneme and accessory elements called outer dense fibers (ODFs). A poorly characterized network of cytoskeletal filaments called the submitochondrial reticulum lies between the ODFs and the mitochondria. The submitochondrial reticulum copurifies with the outer mitochondrial membrane (OMM), suggesting that they are intimately associated (17, 18). Mitochondria wrap around the cytoskeleton and are in turn ensheathed by the plasma membrane. As a consequence of this arrangement, each mitochondrion has three distinct surfaces (19): one facing the axoneme, one facing the plasma membrane, and one facing neighboring mitochondria. Thin-section electron microscopy (EM) (19) and freeze-fracture EM (20, 21) revealed that each surface is characterized by a unique membrane protein profile. In particular, these studies uncovered an ordered array of particles on the axoneme-facing surface of sperm mitochondria. Notwithstanding the insight gained from these methods, such techniques require harsh sample preparation steps that can distort fine cellular structure and limit achievable resolution (22). As such, the molecular landscape of intermitochondrial and mitochondrial–cytoskeleton contacts in the sperm midpiece remains largely unexplored.Assembly of the mitochondrial sheath occurs late in spermiogenesis and involves an intricately choreographed series of events (23, 24). Initially, spherical mitochondria are broadly distributed in the cytoplasm. Mitochondria are then recruited to the flagellum, forming ordered rows along the flagellar axis. Finally, mitochondria elongate and twist around the axoneme. While our understanding of the molecular details of these processes is cursory at best, studies on gene-disrupted mice have implicated several proteins in mitochondrial sheath morphogenesis. For instance, mice expressing mutant forms of kinesin light chain 3 (KLC3) have malformed midpieces, hinting at a role for microtubule-based transport (25). Other examples are the voltage-dependent anion channels (VDACs), which are highly abundant mitochondrial proteins that mediate transport of metabolites, ions, and nucleotides like ATP across the OMM (26). Male mice lacking VDAC3 are infertile, and their sperm cells have disorganized mitochondrial sheaths (27), so VDACs may also have unappreciated roles in mitochondrial trafficking; indeed, KLC3 binds mitochondria through VDAC2 (25). Similarly, disrupting sperm-specific isoforms of glycerol kinase (GK) leads to gaps in the mitochondrial sheath despite proper initial alignment of spherical mitochondria (28, 29). Mice lacking spermatogenesis-associated protein 19 (SPATA19) (30) and glutathione peroxidase 4 (GPX4) (31, 32) also have structurally abnormal mitochondria.Here, we use cryofocused ion beam (cryo-FIB), milling-enabled cryoelectron tomography (cryo-ET) to image the mitochondrial sheath in mature sperm from three mammalian species. We take advantage of the uniquely multiscale capabilities of cryo-ET to unveil aspects both of the overall organization of the mitochondrial sheath and of the molecular structures important for its assembly. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and to the underlying cytoskeleton through conserved protein arrays on the OMM. These arrays were first described by deep-etch, freeze-fracture EM in guinea pig sperm (20). Here, we resolve the three-dimensional structures of the OMM arrays in a near-native state and at molecular resolution, revealing how they anchor onto the mitochondrial membrane and how they interact with the flagellar cytoskeleton. Subtomogram averaging reveals that the arrays consist of twofold-symmetric, boat-shaped particles anchored on a lattice of OMM pores whose arrangement and dimensions are consistent with VDACs. Proteomics and in-cell, cross-linking mass spectrometry (XL-MS) suggest that the arrays consist of GK-like proteins. Our data thus show that although mitochondrial dimensions and cristae architecture vary across species, the architecture of the mitochondria–cytoskeleton interface is conserved at the molecular level.     

Three-dimensional structure of cytoplasmic dynein bound to microtubules

Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located ≈280 Å away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke.     

An icosahedral algal virus has a complex unique vertex decorated by a spike

Paramecium bursaria Chlorella virus-1 is an icosahedrally shaped, 1,900-Å-diameter virus that infects unicellular eukaryotic green algae. A 5-fold symmetric, 3D reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. The pocket might contain enzymes for use in the initial stages of infection. The unique vertex consists of virally coded proteins, some of which have been identified. Comparison of shape, size, and location of the spike with similar features in bacteriophages T4 and P22 suggests that the spike might be a cell-puncturing device. Similar asymmetric features may have been missed in previous analyses of many other viruses that had been assumed to be perfectly icosahedral.     

F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F₁ ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F₁, or the Atp11p and Atp12p chaperones that promote F₁ assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. F₁ mutants are also unable to express ARG8^m when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by F₁ is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of F₁. These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by F₁ to achieve a balanced output of two compartmentally separated sets of ATP synthase genes.     

From the Cover: PNAS Plus: Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

The ability to acquire large-scale recordings of neuronal activity in awake and unrestrained animals is needed to provide new insights into how populations of neurons generate animal behavior. We present an instrument capable of recording intracellular calcium transients from the majority of neurons in the head of a freely behaving Caenorhabditis elegans with cellular resolution while simultaneously recording the animal’s position, posture, and locomotion. This instrument provides whole-brain imaging with cellular resolution in an unrestrained and behaving animal. We use spinning-disk confocal microscopy to capture 3D volumetric fluorescent images of neurons expressing the calcium indicator GCaMP6s at 6 head-volumes/s. A suite of three cameras monitor neuronal fluorescence and the animal’s position and orientation. Custom software tracks the 3D position of the animal’s head in real time and two feedback loops adjust a motorized stage and objective to keep the animal’s head within the field of view as the animal roams freely. We observe calcium transients from up to 77 neurons for over 4 min and correlate this activity with the animal’s behavior. We characterize noise in the system due to animal motion and show that, across worms, multiple neurons show significant correlations with modes of behavior corresponding to forward, backward, and turning locomotion.How do patterns of neural activity generate an animal’s behavior? To answer this question, it is important to develop new methods for recording from large populations of neurons in animals as they move and behave freely. The collective activity of many individual neurons appears to be critical for generating behaviors including arm reach in primates (1), song production in zebrafinch (2), the choice between swimming or crawling in leech (3), and decision-making in mice during navigation (4). New methods for recording from larger populations of neurons in unrestrained animals are needed to better understand neural coding of these behaviors and neural control of behavior more generally.Calcium imaging has emerged as a promising technique for recording dynamics from populations of neurons. Calcium-sensitive proteins are used to visualize changes in intracellular calcium levels in neurons in vivo which serve as a proxy for neural activity (5). To resolve the often weak fluorescent signal of an individual neuron in a dense forest of other labeled cells requires a high magnification objective with a large numerical aperture that, consequently, can image only a small field of view. Calcium imaging has traditionally been performed on animals that are stationary from anesthetization or immobilization to avoid imaging artifacts induced by animal motion. As a result, calcium imaging studies have historically focused on small brain regions in immobile animals that exhibit little or no behavior (6).No previous neurophysiological study has attained whole-brain imaging with cellular resolution in a freely behaving unrestrained animal. Previous whole-brain cellular resolution calcium imaging studies of populations of neurons that involve behavior investigate either fictive locomotion (3, 7), or behaviors that can be performed in restrained animals, such as eye movements (8) or navigation of a virtual environment (9). One exception has been the development of fluorescence endoscopy, which allows recording from rodents during unrestrained behavior, although imaging is restricted to even smaller subbrain regions (10).Investigating neural activity in small transparent organisms allows one to move beyond studying subbrain regions to record dynamics from entire brains with cellular resolution. Whole-brain imaging was performed first in larval zebrafish using two-photon microscopy (7). More recently, whole-brain imaging was performed in Caenorhabditis elegans using two-photon (11) and light-field microscopy (12). Animals in these studies were immobilized, anesthetized, or both and thus exhibited no gross behavior.C. elegans’ compact nervous system of only 302 neurons and small size of only 1 mm make it ideally suited for the development of new whole-brain imaging techniques for studying behavior. There is a long and rich history of studying and quantifying the behavior of freely moving C. elegans dating back to the mid-1970s (13, 14). Many of these works involved quantifying animal body posture as the worm moved, for example as in ref. 15. To facilitate higher-throughput recordings of behavior, a number of tracking microscopes (16–18) or multiworm imagers were developed (19, 20) along with sophisticated behavioral analysis software and analytical tools (21, 22). Motivated in part to understand these behaviors, calcium imaging systems were also developed that could probe neural activity in at first partially immobilized (23) and then freely moving animals, beginning with ref. 24 and and then developing rapidly (17, 18, 25–29). One limitation of these freely moving calcium imaging systems is that they are limited to imaging only a very small subset of neurons and lack the ability to distinguish neurons that lie atop one another in the axial direction of the microscope. Despite this limitation, these studies, combined with laser-ablation experiments, have identified a number of neurons that correlate or affect changes in particular behaviors including the AVB neuron pair and VB-type motor neurons for forward locomotion; the AVA, AIB, and AVE neuron pairs and VA-type motor neurons for backward locomotion; and the RIV, RIB, and SMD neurons and the DD-type motor neurons for turning behaviors (17, 18, 25, 26, 28, 30, 31). To move beyond these largely single-cell studies, we sought to record simultaneously from the entire brain of C. elegans with cellular resolution and record its behavior as it moved about unrestrained.     

Conversion of a yeast prion protein to an infectious form in bacteria

Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [PSI⁺] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology.     

A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin

Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity.     

Development of the malaria parasite in the skin of the mammalian host

The first step of Plasmodium development in vertebrates is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. Here, we document the transformation of sporozoites of rodent-infecting Plasmodium into merozoites in the skin of mice. After mosquito bite, ～50% of the parasites remain in the skin, and at 24 h ～10% are developing in the epidermis and the dermis, as well as in the immunoprivileged hair follicles where they can survive for weeks. The parasite developmental pathway in skin cells, although frequently abortive, leads to the generation of merozoites that are infective to erythrocytes and are released via merosomes, as typically observed in the liver. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist.     

Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis

Alkylresorcinols and alkylpyrones, which have a polar aromatic ring and a hydrophobic alkyl chain, are phenolic lipids found in plants, fungi, and bacteria. In the Gram-negative bacterium Azotobacter vinelandii, phenolic lipids in the membrane of dormant cysts are essential for encystment. The aromatic moieties of the phenolic lipids in A. vinelandii are synthesized by two type III polyketide synthases (PKSs), ArsB and ArsC, which are encoded by the ars operon. However, details of the synthesis of hydrophobic acyl chains, which might serve as starter substrates for the type III polyketide synthases (PKSs), were unknown. Here, we show that two type I fatty acid synthases (FASs), ArsA and ArsD, which are members of the ars operon, are responsible for the biosynthesis of C(22)-C(26) fatty acids from malonyl-CoA. In vivo and in vitro reconstitution of phenolic lipid synthesis systems with the Ars enzymes suggested that the C(22)-C(26) fatty acids produced by ArsA and ArsD remained attached to the ACP domain of ArsA and were transferred hand-to-hand to the active-site cysteine residues of ArsB and ArsC. The type III PKSs then used the fatty acids as starter substrates and carried out two or three extensions with malonyl-CoA to yield the phenolic lipids. The phenolic lipids in A. vinelandii were thus found to be synthesized solely from malonyl-CoA by the four members of the ars operon. This is the first demonstration that a type I FAS interacts directly with a type III PKS through substrate transfer.     

Interplay of Mg2+, ADP,and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg²⁺ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using ³¹P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg²⁺ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg²⁺ in the mitochondrial matrix, where [Mg²⁺] is tenfold higher. In contrast, owing to a much higher affinity for Mg²⁺, ATP is mostly complexed by Mg²⁺ in both compartments. Mg²⁺ starvation used to alter cytosolic and mitochondrial [Mg²⁺] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg²⁺ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.In heterotrophic and well-oxygenated plant cells, ATP is regenerated from ADP principally by glycolysis and mitochondrial oxidative phosphorylation. Surprisingly, although ATP synthesis mechanisms have been deciphered for decades, whether cell respiration is controlled by [ATP]/[ADP] or [ATP]/[ADP][Pi] ratios (1, 2), by the adenylate energy charge ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) (3, 4), and/or by the concentration of ATP or ADP in the cytosol (5, 6) remains a matter of debate. To our knowledge, the determining factor for controlling cell respiration in response to the energy demand has not yet been unambiguously characterized.MgATP is the substrate of numerous phosphorylating enzymes and the principal energy source of the cell. Indeed, any increase in metabolic activity increases the rate of MgATP use and, consequently, the rate of ADP and magnesium release, and vice versa. In normoxia, the MgATP concentration should be essentially balanced by the ADP phosphorylation catalyzed by mitochondrial ATP synthase, thereby adjusting oxidative phosphorylation to cell ATP needs. The ADP/ATP carrier (AAC) of the inner mitochondrial membrane, which exchanges free nucleotides, and adenylate kinase (EC 2.7.4.3), which interconverts MgADP and free ADP with MgATP and free AMP in the presence of Mg²⁺ (7), participate in this regulation (reviewed in ref. 8). Clearly, to better understand the interplay of free and Mg-complexed ADP and ATP in the regulation of cell respiration it is necessary to know their concentrations, as well as the concentration of Mg²⁺ in the cytosol and mitochondrial matrix.Nucleotides can be measured using ³¹P-NMR spectroscopy both in vitro, from cell extracts, and in vivo, in perfused material. After 1 h of data accumulation time, detection thresholds are approximately 20 nmol in vitro and 50 nmol in vivo (9). Various techniques for measuring intracellular [Mg²⁺] and free/Mg-complexed nucleotides have been proposed (10–12), but none allows measurement in different intracellular compartments. In vivo ³¹P-NMR spectroscopy offers this possibility, because the chemical shift (δ) of the γ- and β-phosphorus resonances of ATP and the β-phosphorus resonance of ADP depend on pH and [Mg²⁺] (13). We adapted this noninvasive technique to the simultaneous in vivo measurement of cytosolic and mitochondrial Mg²⁺ and free/Mg-complexed nucleotides concentrations in culture cells.We used homogenous cells cultivated on liquid nutrient media (NM) so as to narrow resonance peaks on in vivo NMR spectra, thus improving the signal-to-noise ratios and the accuracy of chemical shift measurements and limiting peak overlaps. In addition, the heterotrophic sycamore (Acer pseudoplatanus L.) cells of cambial origin used in this study contain no large chloroplasts, but only small plastids (14, 15) with low amounts of nucleotides (16), thus permitting more precise measurement of the cytosolic and mitochondrial nucleotide pools.To modify nucleotide concentrations without using inhibitors that may interfere with mitochondrial functioning, we varied the cell culture media: standard, adenine-supplied, Pi-starved, and Mg-starved. In this paper, we refer to cytoplasm as the cell compartment exterior to the vacuole and cytosol as the cell compartment exterior to the vacuole and the organelles bounded by a double membrane (mitochondria and plastids).The aim of the present study was to determine the role of ADP, ATP, and Mg²⁺ concentrations in the in vivo control of mitochondrial respiration. We show that the balance between cytosolic and mitochondrial free ADP, depending on the concentration of Mg²⁺ in the cytosol and matrix, mediates this regulation.     

Analysis of cell division patterns in the Arabidopsis shoot apical meristem

The stereotypic pattern of cell shapes in the Arabidopsis shoot apical meristem (SAM) suggests that strict rules govern the placement of new walls during cell division. When a cell in the SAM divides, a new wall is built that connects existing walls and divides the cytoplasm of the daughter cells. Because features that are determined by the placement of new walls such as cell size, shape, and number of neighbors are highly regular, rules must exist for maintaining such order. Here we present a quantitative model of these rules that incorporates different observed features of cell division. Each feature is incorporated into a “potential function” that contributes a single term to a total analog of potential energy. New cell walls are predicted to occur at locations where the potential function is minimized. Quantitative terms that represent the well-known historical rules of plant cell division, such as those given by Hofmeister, Errera, and Sachs are developed and evaluated against observed cell divisions in the epidermal layer (L1) of Arabidopsis thaliana SAM. The method is general enough to allow additional terms for nongeometric properties such as internal concentration gradients and mechanical tensile forces.The Arabidopsis shoot apical meristem (SAM) is a structure at the tip of the shoot that is responsible for generating almost all of the above-ground tissue of the plant (1). Its epidermal and subepidermal cells are organized into layers with very few cells moving between layers (2, 3). When these cells expand they do so laterally, pushing other cells toward the periphery of the meristem. Division in these cells is anticlinal such that each layer remains one cell thick. The underlying mechanism determining the location of new cell walls is unknown but the qualitative properties of meristematic cell division are well documented (4–8). Perhaps the best known summary is Errera’s rule, derived following observations of soap bubble formation. In the modern interpretation, the plane of division corresponds to the shortest path that will halve the mother cell. Errera, in fact, wrote that the wall would be a surface “mit constanter mittlerer Krümmung (= Minimalfläche) [with constant mean curvature (= minimal area)]” (4). Because this does not specify a location for the new cell wall, more recent authors have added to this that the mother cell divides evenly (9, 10). With this modification, Errera’s rule is easily quantifiable.A second observation is Hofmeister’s rule: New cell walls usually form in a plane normal to the principal axis of cell elongation (5). This rule is more difficult to quantify, because the principal axis of cell elongation is often confused with the direction of growth. Cells are asymmetrical and hence a principal direction of cell elongation can easily be calculated (e.g., the principal axis of inertia or principal component of a segmentation). The assumption is often made that because the cell is more elongated in one direction that the primary growth of the cell has been along that direction, but this is not necessarily the case, because the elongation may be derived from a prior cell division. For example, if a symmetrical square divides into two rectangular cells, this does not mean that the two daughter cells have grown primarily along their longer axis. Quantification of cell growth direction is much more difficult: It requires the observation of matching points over time and varies with the internal and external tensile forces on the cell. It is not clear whether the instantaneous direction of cell growth or the longer-term average (e.g., as measured over a significant fraction of a cell generation) is more directly relevant to forming the division plane. Under compression, single cells tend to divide in a plane perpendicular to the principal axis of the stress tensor (11), which could indicate a mechanical basis for cell wall placement.Other observations are that new cell walls form in a plane perpendicular to existing cell walls (6), that cell walls tend to avoid four-way junctions (7), and that cell division planes tend to be staggered, like bricks in a wall (8). Because chemical signals can be induced by physical interactions such as mechanical stress and strain it is conceivable that these geometric indicators are merely emergent properties of the underlying physicochemical interaction processes that drives cell division. Although most of the geometric observations tend to be true most of the time, none of them is true all of the time, and it is not possible for all of them to be true at once. For example, the actual growth direction is rarely in alignment with the principal geometric axis of the cell, and hence the division cannot simultaneously satisfy shortest length and perpendicularity requirements. Such conflicting results can in principle be resolved by minimizing a sum of potential functions (12), and insight can often be gained into the underlying mechanisms by examining the results of the optimization. Additionally, recent work by Besson and Dumais (9) suggests that cell division in plants is inherently random. The new wall tends to find a global minimum length, but in situations where there are multiple similar local minima the global minimum is not necessarily chosen.Previously we looked at cell divisions in the shoot meristem using 2D maximum intensity projections (13). Some of the results from that work may have been biased owing to the inconsistent perspective on cells in the peripheral zone compared with the center created by projecting a 3D object into 2D space. Because the meristem is dome-shaped, when projecting the meristem from the top the cells in the center are viewed perpendicularly, whereas the cells toward the edges are viewed at an angle. This nonperpendicular viewing angle distorts the lengths of the cell walls and the angles at which the walls join each other. To rectify that problem the geometry of the cells must be examined in 3D. Here we expand on earlier work with more comprehensive 3D image processing techniques to analyze the division patterns in the local tangent plane. By using the image processing software MorphoGraphX (14) we were able to reconstruct the cell boundaries in the first layer of a growing SAM.Having a 3D model of the structure of the epidermal (L1) layer over time allowed us to generate a model composed of a set of functions, each incorporating a different feature from the observed cell divisions. The functions each contribute a single term to a greater potential function and new walls are predicted to form where the combined potential is reduced. This model also brought to light some of the shortcomings of previously proposed plant cell division rules. Additionally, these data allowed us to make the observations reported below of the dynamics of cell expansion and division in different regions of the SAM.     

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [⁶⁴Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [⁶⁴Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [¹⁸F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.Despite the success of therapeutics fighting against especially bacteria and fungi, infectious diseases still remain one of the main causes of death worldwide (1). Beside effective therapeutics, the early and reliable differential diagnosis of infectious diseases is of utmost importance; here noninvasive imaging can have a huge impact. Imaging of infectious diseases is an emerging field still in its infancy, but is nevertheless attracting considerable attention from many disciplines in biomedical research, as well as in patient care. There are several challenging aspects of imaging infectious diseases, not at least the clear and reliable differentiation between bacterial, fungal, and viral infection needed for the best treatment options. Furthermore, infection is typically linked to inflammation, which makes it mandatory to use pathogen specific imaging probes to definitively and rapidly diagnose the causative agent of the infectious disease.Invasive pulmonary aspergillosis (IPA) is a frequently fatal lung disease of neutropenic patients caused by the ubiquitous airborne fungus Aspergillus fumigatus. As a leading cause of death in hematological malignancy and hematopoietic stem cell transplant patients, the fungus accounts for the majority of the >200,000 life-threatening infections annually with an associated mortality rate of 30–90% (2). Diagnosis of IPA is a major challenge as clinical manifestations of the disease (febrile episodes unresponsive to antibiotics, pulmonary infiltrates and radiological abnormalities) are nonspecific, and methods for the detection of circulating biomarkers such as β-d-glucan or galactomannan (GM) in the bloodstream lack specificity or sensitivity (3). For this reason, culture of the fungus from lung biopsy tissues remains the gold standard test for IPA diagnosis (4), but this invasive procedure lacks sensitivity, delays diagnosis, and is frequently not possible in neutropenic patients. Recently, detection of A. fumigatus GM or mannoprotein antigens in bronchoalveolar lavage (BAL) has shown enormous promise for the early detection of the disease especially when combined with point-of-care diagnostics (5). However, BAL recovery is similarly intrusive and so a sensitive, specific, and minimally invasive test that is amenable to repeated application is needed to allow diagnostic-driven treatment with antifungal drugs. Such a test should be able to discriminate between active lung infection caused by hyphal proliferation of the fungus and inactive spores that are a common component of inhaled air. Conventional imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI) are able to produce high contrast images of all structures within the human body but they are not able to distinguish between invasive fungal infections and those caused by other microorganisms, or to discriminate these from cancer tissues (6, 7). Molecular imaging using positron emission tomography (PET) is able to define the metabolic properties of living cells as well as their molecular structures when suitable radiolabeled tracers are used (8). Here, we use a radiolabeled monoclonal antibody (mAb) specific to the active growth phase of A. fumigatus to diagnose IPA in a neutropenic animal model of the disease with PET/MRI. Our work shows that antibody-based immunoPET can be used successfully to noninvasively identify this challenging lung disease.