Search

Motor neurons are a remarkably powerful cell type in the central nervous system. They innervate and control the contraction of virtually every muscle in the body and their dysfunction underlies numerous neuromuscular diseases. Some motor neurons seem resistant to degeneration whereas others are vulnerable. The intrinsic heterogeneity of motor neurons in adult organisms has remained elusive. The development of high-throughput single-cell transcriptomics has changed the paradigm, empowering rapid isolation and profiling of motor neuron nuclei, revealing remarkable transcriptional diversity within the skeletal and autonomic nervous systems. Here, we discuss emerging technologies for defining motor neuron heterogeneity in the adult motor system as well as implications for disease and spinal cord injury. We establish a roadmap for future applications of emerging techniques - such as epigenetic profiling, spatial RNA sequencing, and single-cell somatic mutational profiling to adult motor neurons, which will revolutionize our understanding of the healthy and degenerating adult motor system.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1–6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system. Astrocytes can respond to diseases and injuries of the central nervous system by driving the death of neurons and mature oligodendrocytes through the delivery of long-chain saturated fatty acids contained in lipoparticles.

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease. Blum et al. performed single-nucleus RNA sequencing of the adult mouse spinal cord. This analysis revealed heterogeneity in the autonomic and skeletal motor systems and provides a resource to study motor neurons in health and disease.

SummaryAntisense oligonucleotide therapy targeting ATXN2—a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis—has entered clinical trials in humans. Additional methods to lower ataxin-2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin-2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v-ATPase) as regulators of ataxin-2 levels. We demonstrate that Etidronate—a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v-ATPase—lowers ataxin-2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.

Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease

The spinal cord is a fascinating structure responsible for coordinating all movement in vertebrates. Spinal motor neurons control the activity of virtually every organ and muscle throughout the body by transmitting signals that originate in the spinal cord. These neurons are remarkably heterogeneous in their activity and innervation targets. However, because motor neurons represent only a small fraction of cells within the spinal cord and are difficult to isolate, the full complement of motor neuron subtypes remains unknown. Here we comprehensively describe the molecular heterogeneity of motor neurons within the adult spinal cord. We profiled 43,890 single-nucleus transcriptomes using fluorescence-activated nuclei sorting to enrich for spinal motor neuron nuclei. These data reveal a transcriptional map of the adult mammalian spinal cord and the first unbiased characterization of all transcriptionally distinct autonomic and somatic spinal motor neuron subpopulations. We identify 16 sympathetic motor neuron subtypes that segregate spatially along the spinal cord. Many of these subtypes selectively express specific hormones and receptors, suggesting neuromodulatory signaling within the autonomic nervous system. We describe skeletal motor neuron heterogeneity in the adult spinal cord, revealing numerous novel markers that distinguish alpha and gamma motor neurons—cell populations that are specifically affected in neurodegenerative disease. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neurons. Collectively, these data provide a single-cell transcriptional atlas for investigating motor neuron diversity as well as the cellular and molecular basis of motor neuron function in health and disease.

Microglia are the resident macrophages of the CNS. Though they are best known for their traditional macrophage activities, such as the phagocytosis of cell debris and inflammatory responses after injury, microglia also play an integral role in the normal development of the brain and spinal cord. For

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Crosses betweenDrosophila melanogasterfemales andDrosophila simulansmales produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in theD. melanogaster Hybrid male rescue(Hmr) or in theD. simulans Lethal hybrid rescue(Lhr)genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements withinD. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains fromHmrandLhrsingle mutants andHmr; Lhrdouble mutants inD. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not localize to centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin-heterochromatin junction. The few dividing cells present in hybrid males showed irregularly condensed chromosomes with fuzzy and often closely apposed sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr withinD. melanogasternor for a centromere defect causing hybrid lethality. Instead we find thatHmrandLhrare required inD. melanogasterfor detachment of sister chromatids during anaphase.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

In two previous papers [Reed and Blum, J. Acoust. Soc. Am. 97, 425-438 (1995), Blum et al., J. Acoust. Soc. Am. 98, 181-191 (1995)] a computational model for signal processing in the dorsal cochlear nucleus (DCN) was developed. In those modelling studies, stellate cells inhibited only type II cells. In this study, the effect of including wide-band inhibitory (WBI) connections from stellate cells to type IV cells, as proposed by Nelken and Young [J. Neurophysiol. 71, 2446-2462 (1994)], is examined. Inclusion of the WBI connections improves the fit to the experimental pure tone response maps for both the "standard" and "non-standard" cells examined by Spirou and Young [J. Neurophysiol. 66, 1750-1768 (1991)]. Thus, these modelling studies support the hypothesis of Nelken and Young [J. Neurophysiol. 71, 2446-2462 (1994)]. The degree of improvement is greatest for cells with prominent upper inhibitory sidebands. The qualitative features of the pure tone response map and the theoretical model allow one to deduce the probable frequency bias of the type II to type IV and stellate to type IV connections.

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2–100 μm, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μm. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nm) to very low (>100 μm) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

A structural model is proposed for the processing of interaural intensity differences by the lateral superior olive. One fundamental assumption is that the incoming excitatory projections from the ipsilateral anteroventral cochlear nucleus innervate columns of LSO neurons serially according to threshold. A second fundamental assumption is that the inhibitory innervation from the ipsilateral medial nucleus of the trapezoid body is also serially arranged according to threshold but in the opposite direction along the LSO column. Using neurophysiological and neuroanatomical data for neuronal response curves, connectional patterns, and cell and synapse numbers, the model was formulated quantitatively and implemented for machine computation. Azimuthal location is encoded by the position along the LSO column where LSO cell firing first goes to zero. Accuracy of coding was tested for three different connectional schemes, for variations in neuronal parameters, and for cell and synapse death. Encoding is shown to be independent of absolute sound level and to vary linearly with interaural intensity difference.

Linear reaction-hyperbolic equations of a general type arising in certain physiological problems do not have traveling wave solutions, but numerical computations have shown that they possess approximate traveling waves. Using singular perturbation theory, it is shown that as the rates of the chemical reactions approach $\infty $, solutions approach traveling waves. The speed of the limiting wave and the first term in the asymptotic expansion are computed in terms of the underlying chemical mechanisms.

In a previous modeling study of signal processing in the dorsal cochlear nucleus [Reed and Blum, J. Acoust. Soc. Am 96, 1442-1453 (1997)] it was shown that inclusion of a wideband inhibitor (WBI) greatly improved the fit between model response maps and the experimental response maps of type IV units to pure tones. In this study we examine the effect of the WBI on the responses to complex sound stimuli such as broadband noise (BBN), notch noise, noise bands, and band/notch combinations. A new and more realistic model for auditory nerve (AN) response in the presence of different levels of noise is used. It is shown that one can explain and understand the qualitative features of virtually all the published data on type II and type IV unit responses to BBN, notch noise and noise bands. The monotone decreasing response of the maximum firing rate of type II units to noise bands of increasing width that is observed experimentally occurs in the model due to the increasing inhibition of type II cells by the WBI. Similarly, the various nonmonotone patterns of maximum firing rate of type IV units to noise bands of increasing width is shown to arise from the complex and highly nonlinear effects of inhibition from the type II to type IV and the WBI to type IV cells and the nonlinear direct excitation from the AN to the type IV cells. A number of experiments using double notches, double noise bands, or notch-noise band pairs are suggested which, by comparison with model results, would allow one to infer probable connectional patterns between type II and type IV units and between the WBI units and the type IV units.

In a previous paper a computational model was developed which was shown to account for most of the essential features of the variety of experimentally observed response maps of type‐IV cells in the dorsal cochlear nucleus to pure tones. In the present study, the responses of the same model DCN to broadband noise and notch noise are investigated. It is shown that the previous model qualitatively accounts for the observed responses to these more complex sounds. Predictions of the model for inverted notches and for the behavior of type‐IV cell output as notch center is varied for different amplitudes are presented. It is shown that the model is suitable for feature detection of auditory signals and an expansion is given as to how this ability arises from the properties of the stellate, type‐II, and type‐IV cells and the variations in the connectional patterns that were previously shown to account for the response patterns of type‐IV cells to pure tones.

Much information is available on the anatomical organization and neurophysiological properties of the major cell types in the dorsal cochlear nucleus (DCN). The complicated response properties of individual cells and units in the DCN indicate that substantial information processing already occurs at the level of the DCN. A large number of connectional hypotheses have been put forward to explain various aspects of the response characteristics of DCN cells, but many of the consequences of these hypotheses have not been investigated quantitatively. In this paper, we investigate these hypotheses by constructing and testing mathematical and computational models and compare our results to those of previous modeling studies. The simplest versions of our models include auditory nerve (AN) fibers, type II cells (inhibitory interneurons) and type IV cells (fusiform and giant cells). The model response maps, i.e., the pattern of output of model type IV cells, generated by the simplest model have some but not all the features of the experimental response maps of type IV neurons. In particular, the excitatory region which occurs at best frequency is not isolated and the excitatory region at low frequencies and high amplitude is narrower than observed. Since experimental evidence exists that some of the connections between these cell types are divergent or convergent across adjacent isofrequency sheets, the effect of such convergence and divergence was then investigated. Response maps so obtained reproduce many of the qualitative features of the experimental maps. Enlargement of the model by including inhibitory interneurons (e.g., stellate cells) that receive convergent input from AN fibers and which inhibit type II cells results in the generation of response maps that, with some variations in connectional patterns and physiological properties of neurons, match most of the essential features seen in the large variety of experimental maps obtained from the cat DCN.

A number of investigators have published measurements of the outputs of single neurons from the lateral superior olive (LSO) of the cat for a variety of auditory signals. The response curves show a very wide range of shapes and thresholds. In this paper, the single neuron response curves predicted by a previously published model for the encoding of azimuthal location by the LSO to the experimental curves are compared. The predicted curves are in good qualitative agreement with the experiments and, in addition, the model provides an explanation of the seemingly paradoxical drop in output as interaural intensity difference (IID) is held fixed and absolute intensities are raised. The particular shape of the response curve depends on the location of the LSO neuron examined. In the model, two possible developmental programs to form the adult pattern of connections from the anteroventral cochlear nucleus and the medial nucleus of the trapezoid body onto the LSO are also examined. In the first, connections are made by a forward stochastic process in which there is a limit on the numbers of synapses formed on each LSO cell. In the second, there is no such limit, but in later developmental stages pruning of synapses occurs which reduces their number to the limit. Both schemes give similar adult connectional patterns. The LSO response curves generated by the model are similar to those observed in LSO neurons of the developing gerbil by Sanes and Rubel [J. Neurosci. 8, 682–700 (1988)]. Thus the model mechanism not only encodes azimuthal location by activity across the population of neurons, but is also consistent with single unit neurophysiological measurements of LSO output in both developing and adult animals.

The compartmentalization of acetyl-CoA metabolism in Tetrahymena has been studied by measuring the incorporation of label from [1-14C]acetate, [2-14C]pyruvate, and [1-14C]-octanoate into CO2, lipids, glycogen, and glutamate in experiments in which these substrates were used singly, in pairs, and all together. The results of these studies require that there be at least three pools of acetyl-CoA in Tetrahymena, two in the mitochondria and one in the peroxisomes. A steady state model was developed which allows a quantitative analysis of the metabolic fate of acetyl-CoA derived from acetate, pyruvate, and octanoate. The model shows that pyruvate enters a different mitochondrial pool of acetyl-CoA than acetate or octanoate, which accounts for the relatively high proportion of [2-14C]pyruvate label incorporated into glycogen. About three-fourths of the acetate utilization occurs in the mitochondria, the remainder via the peroxisomal pool of acetyl-CoA for lipogenesis. Only part of the octanoate utilization is accounted for by β oxidation in the peroxisomes; a pathway is also required allowing the incorporation of label from octanoate into lipids without prior oxidation to acetyl-CoA. Labeled acetyl-CoA used for glyconeogenesis comes from both the peroxisomal and the outer mitochondrial pools of acetyl-CoA, more of it coming from the former. The experiments were performed on well aerated cultures in both the logarithmic and transitional phases of growth. Differences in metabolic flux patterns between cells at these two stages of growth are small; lipogenesis was decreased and glutamate output and pyruvate utilization were increased in transition as compared to log cultures.

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++-dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.

Tetrahymena were grown with shaking for 17 hours in the presence and absence of AMP. Under these conditions, glycogen content increased but there was a marked inhibition of glyoxylate oxidation and of the incorporation of label from acetate into glycogen during a 1-hour incubation. AMP treatment also caused and increase in the rate of incorporation of [3H]uracil into RNA which was maximal about 2 hours after the start of growth. Experiments were performed in which cells from log and transition phase cultures were incubated with acetate, pyruvate, and octanoate, but with only one substrate labeled in any one flask. Measurements were made of the amount of label incorporated from each labeled substrate into glycogen, lipids, CO2, and glutamate, and the results were analyzed according to a previously established model for the structure of metabolism in Tetrahymena. It was found that the model, which utilizes three pools of acetyl-CoA, permitted a quantitative fit to the data. Analysis of the data showed that AMP treatment reduced the rate of the Krebs cycle, the rate of glyconeogenesis, and the rate of pyruvate utilization in both log and transition phase cells from these labeled substrates. Incorporation of label from acetate into glutamate, however, was increased by AMP treatment in log cells and decreased in transition phase cells. AMP treatment markedly decreased the fraction of label entering into glyconeogenesis from the peroxisomal pool of acetyl-CoA. There was no change in the ATP:ADP ratio in log cells treated with AMP, and a slight decrease in the sum ATP + ADP. In transition phase cells, however, the ATP:-ADP ratio decreased and the sum of ATP + ADP also increased compared to control cells. Since many of the changes in intracellular flux patterns occurred in both log and transitional cells, changes in enzyme levels or control mechanisms other than the ATP:ADP ratios must be invoked to explain the effects of AMP on metabolism.

A metabolic scheme of glycolysis and the pentose phosphate pathway has been constructed, assuming that the reactions occur in a single compartment. From this scheme, equations are written for a system in metabolic and isotopic steady state. These allow computation of the specific activity of every carbon atom of all the intermediates of the glycolytic and pentose phosphate pathways and consequently of the flux of carbon along each step of these pathways. A sufficiently large number of well distributed measurements of incorporation of radioactive label from different positions of several substrates into intermediates or products must be made to determine all the fluxes. This is done by choosing a set of metabolic fluxes, calculating incorporation with the aid of a computer, and then manipulating the flux rates until the computed incorporations match the data. The model is used in this paper to analyze the metabolism of the protozoan Tetrahymena pyriformis. The metabolic scheme of the model is consistent with all available information on the enzyme complement of this ciliate. Cells grown to transition phase in proteose/peptone medium were inoculated into a mixture of glucose (6 mM), fructose (6 mM), ribose (3 mM), and glycerol (3 mM) and incubated for 1 h. In each of these experiments, one of the following labeled substrates was present: [1-, 2-, 6-, or U-14C]glucose; [1- or U-14C]fructose; [1- or U-14C]ribose; [1(3)-or 2-14C]glycerol. The incorporation of label from these substrates into CO2, lipid, glycogen, and RNA was measured. In contrast to earlier studies on the metabolism of 2- and 3-carbon substrates by Tetrahymena, the rate of incorporation of label from some substrates into some products (e.g. from [1-14C]glucose into CO2) changed during the incubation. To treat these time-dependent data within the framework of the steady state model, the 1-h incubation was divided into three 20-min intervals; within each of these, the rates of incorporation were approximately constant, as required for a steady state system. Measurements of the pool sizes of glucose-6-P and fructose-6-P showed that only slow changes in pool sizes occurred after the first 5 min of incubation and indicated that the system was effectively in a metabolic and isotopic steady state throughout most of the incubation. The finding that a low concentration of cycloheximide prevented the acceleration of 14CO2 production from labeled glucose suggests a role for protein synthesis in the slow adaptation to carbohydrate addition and supports the quasi-steady state treatment of this system. The expected incorporation into each product was computed for trial sets of 1, independent flux rates. A set of flux values was found which yielded a good fit to the 29 measurements made for each interval. These flux values therefore constitute a quantitative description of temporal changes in carbon flow along the glycolytic and pentose phosphate pathways during the 1st h of adaptation to the carbohydrate mixture...

Cultures of Tetrahymena pyriformis were incubated for 1 hour with a mixture of acetate, pyruvate, and pentanoate with only one substrate labeled at a time and with the position of the label chosen so that [1-14-C]acetyl coenzyme A was an early product of the metabolism of each substrate. The appearance of label in CO2, lipids, glycogen, glutamate, and alanine were measured and results interpreted in terms of a previously developed three-compartment model of metabolism, which was found to quantitatively describe the data even when two of the flux rates (the flux of acetyl-CoA from the peroxisomal to the outer mitochondrial compartment and from the outer mitrochondrial to the inner mitochondrial compartment) were set equal to zero. This reduction in the number of independent parameters leads to the model being overdetermined and to a probably unique fit of the three-compartment model tof the present data and to previous data when octanoate was the fatty acid substrate. Pentanoate was metabolized to a greater extent than octanoate and did not inhibit growth. Pentanoate inhibited acetate utilization in both the inner mitochondrial and peroxisomal compartments as indicated by a reduction in the incorporation of label from [1-14-C]acetate into lipids and into CO2, but there was no difference in oxidation of [2-14-C]pyruvate when pentanoate was the fatty acid substrate as compared to octanoate. Glyconeogenesis was inhibited when pentanoate was substituted for octanoate. Similar experiments were performed on cells treated with 4-pentenoic acid. The effects of 4-pentenoic acid were essentially the same whether octanoate or pentanoate was the fatty acid substrate, i.e. inhibition of glyconeogenesis from all labeled substrates and inhibition of [2-14-C]pyruvate oxidation. The results indicate that the effects of pentanoate are largely confined to the peroxisomal and the inner mitochondrial compartments whereas the effects of 4-pentenoic acid are confined to the peroxisomal and outer mitochondrial compartments.

Log-phase Tetrahymena were washed and resuspended in a dilute salt solution supplemented with glucose, acetate, pyruvate, or carmine, as desired, and then incubated for 5 h. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed. Extracellular activities were corrected for proteolytic degradation. The three nutritive substrates affected both the amount and pattern of extracellular enzyme release, but carmine had no effect. Intracellular activities declined early in the starvation period, but partially recovered with time, particularly α-glucosidase activity. Acetate reduced the decline in acid phosphatase activity; acetate and glucose enhanced the recovery of α-glucosidase activity; carmine had no effect on intracellular enzyme activities. Protein content changed little and was unaffected by the addition of substrates. Glycogen content increased during incubation; acetate and glucose enhanced the increase.

At least three-fourths of the phosphofructokinase activity in homogenates of Tetrahymena pyriformis is localized on the mitochondria. The mitochondrial phosphofructokinase activity is stabilized by ATP and by fructose 6-phosphate and is inhibited by ATP and by citrate.

The incorporation of 14C into CO2 from ten labeled substrates: [1-14C]- and [2-14C]acetate; [1-14C]-, [3,4-14C]-, and [5-14C]glutamate; [1-14C]-, [4-14C]-, and [U-14C]aspartate, [1-14C]- and [U-14C]alanine was measured under steady state conditions in Tetrahymena pyriformis cultures at three different stages of growth. The rate of oxygen consumption and the incorporation of 14C from [1-14C]- and [2-14C]acetate into lipid and glycogen were measured under the same conditions. An isotopic and metabolic steady state model of carbon flow through the tricarboxylic acid cycle, the glyoxylate cycle, and the gluconeogenic and lipogenic pathways in Tetrahymena was developed. Fitting of the model to experimental data requires separation of mitochondrial and extramitochondrial pools of aspartate, oxalacetate, malate, and acetyl-CoA, as well as two pools of phosphoenolpyruvate. These structural requirements, necessary to fit the experimental data, were independently justified by information on enzyme compartmentation. By using the data in conjunction with the model quantitative estimates of the flux rates through the major pathways of intermediary metabolism were obtained. The pattern of intracellular flow of metabolites underlying the observed changes in glycogen content and lipid content with culture age are analyzed.

At moderate concentrations the methyl, ethyl, or butyl esters of 10 amino acids inhibit the growth of Astasia. At least 8 of these also inhibit the growth of Euglena, although Euglena is generally less sensitive to these esters than Astasia. None of the amino acids tested at comparable concentrations inhibit growth except cysteine. The concentrations of ester required for 50% inhibition of growth rate depend not only on the amino acid, but on the ester group, increasing inhibition being generally observed with increasing size of the alcohol moiety. Recovery takes place primarily by nonenzymatic hydrolysis of the ester. Inhibition is highly pH-sensitive, with little inhibition below pH 5 and maximal inhibition above pH 6. Experiments with 14C-leucine ethyl ester indicate that Astasia is much less permeable to this ester at low pH. These observations led to the hypothesis that the amino acid esters inhibited growth by interfering with the corresponding aminoacyl transfer ribonucleic acid (tRNA) synthetases. Consistent with this hypothesis were the observations that: (a) phenylalanine ethyl ester inhibited the synthesis of an induced acid phosphatase; (b) the methyl and ethyl esters of serine, which did not inhibit growth, did not inhibit a partially purified seryl-tRNA synthetase of Astasia; (c) the methyl and ethyl esters of leucine, which inhibited growth, competitively inhibited the partially purified leucyl-tRNA synthetase of Astasia. Other growth experiments demonstrated that, whereas leucine slightly protected against growth inhibition by leucine ethyl ester, phenylalanine potentiated growth inhibition due to phenylalanine ethyl ester. It was then found that partially purified phenylalanyl-tRNA synthetase is inhibited by excess substrate concentrations. It is suggested that some amino acid esters may be useful inhibitors of the first committed steps of protein synthesis.

At low concentrations of ATP glycerinated cilia swell, as indicated by an increase in packed volume after low speed centrifugation. At high concentrations of ATP partial dissolution occurs. The soluble proteins thus obtained were resolved by sucrose density gradient sedimentation. Three major peaks were obtained: a nonadenosine triphosphatase protein sedimenting at 4 S; an ATPase of low specific activity sedimenting at 14 S; and an ATPase of high specific activity and high substrate specificity sedimenting at 30 S. The pellet remaining after extraction contained ciliary membranes and outer fiber pairs, and had ATPase activity of low specific activity and low substrate specificity. After extraction with high ATP the residual pellet no longer swelled in response to the addition of a low concentration of ATP. The 30 S protein can recombine functionally with the pellet, as judged by an increase in turbidity which could be reversed by the addition of ATP. The low specific activity, 14 S ATPase is activated by ethylenediaminetetraacetic acid, but the 30 S ATP is not. Both ATPases are activated by Mg++ and by Ca++. The fractions obtained by this method resemble those obtained by Gibbons from ethanol- and calcium-isolated cilia by his fractionation procedure.

Low concentrations of p -chloromercuribenzoate (PCMB), Zn ++ , or Cd ++ increase the ATPase and CTPase activities of myosin B but inhibit ITPase and GTPase activities. Higher concentrations of these modifiers inhibit the activities of all the nucleoside triphosphates (NTP). Dialysis against cysteine ethyl ester (CEE) increases the ATPase and CTPase activities of myosin but decreases the ITPase and GTPase activities. The temperature dependence of the maximum velocities of hydrolysis and of the Michaelis-Menten constants also differentiate between ATP-CTP and ITP-GTP. ATP and CTP confer considerable protection against thermal denaturation of myosin, while ITP and GTP confer only a small protection. The shape of the PCMB vs. NTPase activity curve is invariant to the substitution of Mg ++ for Ca ++ in the presence of 0.3 M KCl or to the substitution of Na + , Li + , or NH 4 + for K + in the presence of Ca ++ . PCMB inactivates myosin at 37 ° at a rate which is highly sensitive to the PCMB concentration. The inactivated myosin can be restored to activity by dialysis against CEE. These and other observations are interpreted in terms of a conformation change at the active site of myosin.

Mannose inhibited the growth rate of Euglena when glucose, fructose, pyruvate, acetate, ethanol, succinate, oxalacetate, or glutamine was the sole source of carbon. The degree of inhibition was a function of the particular substrate and, in the case of glucose, of the glucose to mannose concentration ratio. The metabolic basis of growth inhibition was investigated. Mannose does not critically inhibit glucose entry or phosphorylation and does not interfere with the Krebs cycle. Mannose 6-phosphate, however, is an inhibitor of phosphoglucose isomerase, and this may account for much of the growth inhibition when fructose or glucose is the carbon source. That inhibition at this site alone cannot account entirely for growth inhibition is indicated by the sensitivity to mannose of growth on substrates which enter metabolism at the level of the Krebs cycle, and by the results of experiments in which pairs of substrates were used as carbon sources. Mannose by itself does not support growth. 14C from mannose-1-14C, however, was incorporated into protein, phospholipids, and wax, but not into CO2. Trapping of 14C-acetyl groups generated from glucose-1-14C with sulfanilamide was associated with a decrease in 14CO2 and 14C-phospholipid, but not of 14C-wax formation, whereas sulfanilamide inhibited neither 14C-phospholipid nor 14C-wax formation from mannose-1-14C. Mannose increased 14CO2 production and 14C incorporation from pyruvate-2-14C into phospholipids, but decreased 14C incorporation into wax. Based on these observations, the existence of two metabolic compartments for lipid synthesis in Euglena is postulated.

Cell-free extracts of Astasia longa possess the following enzymes at the indicated relative activities: (a) quinolinic acid phosphoribosyl transferase, 0.5; (b) nicotinic acid mononucleotide pyrophosphorylase, 1; (c) nicotinic acid dinucleotide pyrophosphorylase, 1; (d) nicotinamide adenine dinucleotide synthetase, 0.003; (e) nicotinamide dinucleotide kinase, 1; (f) nicotinamide deamidase, 1; (g) nicotinic acid methyltransferase, 0.0001. The presence of Enzymes a, c, d, and e, in conjunction with data on the intracellular levels of deamido-NMN, deamido-NAD, NAD, and NADP, establishes this sequence of compounds as the pathway of biosynthesis of pyridine nucleotides in Astasia and indicates that the conversion of deamido-NAD to NAD is a rate-limiting step. Cell-free extracts also catalyzed the degradation and pyrophosphorolysis of deamido-NAD and of NAD, and catalyzed an exchange reaction between nicotinic acid and the nicotinamide moiety of NAD. These reactions, in conjunction with the presence of Enzymes b and f, and with data showing a high rate of turnover of NAD in exponentially growing cultures, indicate the operation of a nicotinic acid salvage pathway in Astasia. The finding that nicotinic acid is an inhibitor of Enzyme a and that quinolinic acid is an inhibitor of Enzyme b suggests a possible means of control of the relative rates of the biosynthetic and the salvage pathways. The finding that deamido-NAD, present in relatively high concentrations in Astasia, is an inhibitor of Enzyme b suggests another possible control mechanism. Growth in the presence of nicotinic acid or quinolinic acid did not lead to repression of either Enzyme a or b. Nicotinamide and high concentrations of nicotinic acid inhibited the growth of Astasia and nicotinamide repressed the level of Enzymes a and b.

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.

1. 1. The effects of 2,4-dinitrophenol (DNP) on the interaction of several nucleoside triphosphates with myosin B and glycerol-treated psoas fibers have been studied at 0.3 M KCl, 10−3M Ca++, pH 7.0, 25.5 °C. 2. 2. The sequence of reciprocal Michaelis-Menten constants, K, is K ( ATP > K ( CTP ) > K ( UTP ) > K ( GTP, ITP ) . The sequence of maximal splitting velocities, V, is V (ITP, GTP) > V (UTP) > V (ATP) > V (CTP). These sequences are unchanged by DNP. 3. 3. For ATP, CTP, and UTP, DNP raises V and lowers K a nearly compensatory way. 4. 4. DNP reduces the inhibition of ITPase activity by ADP, decreases the activation of ATPase activity by EDTA, and inhibits the rate of splitting of tripolyphosphate. 5. 5. The non-enzymic alkaline hydrolysis rates of ATP, UTP, GTP, CTP, and ITP are about equal at pH 8.5, 0.3 M KCl, 100 °C. The addition of Mg++ slightly increases the rate. Ca++ approximately doubles the rate. DNP has no effect. Decreasing the salt concentration increases the rate. 6. 6. DNP decreases the amount of shortening of unloaded psoas fibers. 7. 7. The results have been discussed in terms of a kinetic scheme which assumes that the rate of desorption of the nucleoside diphosphate product of the reaction may be rate limiting under certain conditions.

The localization of induced and constitutive acid phosphatase activity in Euglena was studied by light and electron microscopy, using two different cytochemical methods. Cells grown in high phosphate medium have constitutive acid phosphatase activity in three regions: in the Golgi complex, around the paramylum bodies, and in the peri-reservoir vesicles. Cells that have formed an induced acid phosphatase by exposure to a phosphate-deficient medium have, in addition to the constitutive activity localized exactly as in the uninduced cell, a strong activity in the pellicle. The induced activity is not uniformly distributed over the pellicle, but is localized at the notch of each pellicle complex, near a group of about four fibrils and near a characteristic vesicle of the endoplasmic reticulum. In the cytostome, where fission begins during division, there is an alternation of large and small pellicle complexes, both of which have induced phosphatase activity. A similar alternation is seen over the entire pellicle of dividing cells.