Matrix Compartment Research Articles

Single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum

Single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum

NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in three-dimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin.

Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum

The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals immunopositive for vesicular glutamate transporter (VGluT) 2 were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. By contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both compartments. As axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment.

Heparan sulfate regulates the anabolic activity of MC3T3‐E1 preosteoblast cells by induction of Runx2

The transcription factor Runx2 can be controlled by a number of upstream regulators involved in intracellular signalling, including the activation ERK1/2 signaling by fibroblast growth factor-2 (FGF-2). FGFs interact with their cell surface receptors (FGFRs) through an obligate cross-binding interaction with heparan sulfate proteoglycan (HSPG) co-receptors; exogenous HS sugar chains have been shown to potently modulate changes in cell phenotype depending on the stage of tissue differentiation when the HS is harvested, suggesting that HS chain structure and function varies depending on the stage of cell maturity. This study examined the potential of bone-derived heparan sulfate (HS), harvested from differentiating osteoblasts, for the enhancement of preosteoblast growth and differentiation. HS was harvested from conditioned media, cell surface and matrix compartments of postconfluent (differentiating) MC3T3-E1 osteoblasts and dosed back onto preconfluent MC3T3-E1 cells. We show that HS can increase the expression Runx2, ALP, and OPN in preosteoblast cells, suggesting the potential for exogenous HS to shift cells from proliferative to differentiative phenotypes. In line with their structural differences, only HS released into the media was found to co-stimulate the mitogenic effect of FGF-2, whilst exogenous application of all the HSs together with FGF-2 served to increase the expression of OPN. Only the application of cell surface-derived HS triggered a synergistic increase in FGFR1 expression together with FGF-2, although all three HS preparations could trigger transient increases in PI3K, ERK1/2, and stat3 phosphorylation levels. These findings demonstrate that the compartmentally distinct HS species expressed by differentiating MC3T3-E1 cells act in complex ways to coordinate the extracellular conditions that lead to osteoblast differentiation, with the cell surface species coordinating the FGF response.

Striosomes and mood dysfunction in Huntington's disease

Variable phenotype is common in neurological disorders with single-gene inheritance patterns. In Huntington's disease, mood and cognitive symptoms are variably co-expressed with motor symptoms. There is also variable degeneration of neurons in the two major neurochemical compartments of the striatum, the striosomes and the extrastriosomal matrix. To determine whether the phenotypic variability in Huntington's disease is related to this compartmental organization, we carried out a double-blind study in which we used GABA(A) receptor immunohistochemistry to analyse the status of striosomes and matrix in the brains of 35 Huntington's disease cases and 13 control cases, and collected detailed data on the clinical symptomatology expressed by the patients from family members and records. We report here a significant association between pronounced mood dysfunction in Huntington's disease patients and differential loss of the GABA(A) receptor marker in striosomes of the striatum. This association held for both clinical onset and end-stage assessments of symptoms. The cases with accentuated striosome abnormality further exhibited later onset age, lower disease grade and lower CAG repeat length in the HD gene. We found no independent association, however, between CAG repeat length or age of onset and mood dysfunction. We suggest that variation in clinical symptomatology in Huntington's disease is associated with variation in the relative abnormality of GABA(A) receptor expression in the striosome and matrix compartments of the striatum, and that striosome-related circuits may modulate mood functioning.

Differential synaptology of vGluT2‐containing thalamostriatal afferents between the patch and matrix compartments in rats

The striatum is divided into two compartments named the patch (or striosome) and the matrix. Although these two compartments can be differentiated by their neurochemical content or afferent and efferent projections, the synaptology of inputs to these striatal regions remains poorly characterized. By using the vesicular glutamate transporters vGluT1 and vGluT2, as markers of corticostriatal and thalamostriatal projections, respectively, we demonstrate a differential pattern of synaptic connections of these two pathways between the patch and the matrix compartments. We also demonstrate that the majority of vGluT2-immunolabeled axon terminals form axospinous synapses, suggesting that thalamic afferents, like corticostriatal inputs, terminate preferentially onto spines in the striatum. Within both compartments, more than 90% of vGluT1-containing terminals formed axospinous synapses, whereas 87% of vGluT2-positive terminals within the patch innervated dendritic spines, but only 55% did so in the matrix. To characterize further the source of thalamic inputs that could account for the increase in axodendritic synapses in the matrix, we undertook an electron microscopic analysis of the synaptology of thalamostriatal afferents to the matrix compartments from specific intralaminar, midline, relay, and associative thalamic nuclei in rats. Approximately 95% of PHA-L-labeled terminals from the central lateral, midline, mediodorsal, lateral dorsal, anteroventral, and ventral anterior/ventral lateral nuclei formed axospinous synapses, a pattern reminiscent of corticostriatal afferents but strikingly different from thalamostriatal projections arising from the parafascicular nucleus (PF), which terminated onto dendritic shafts. These findings provide the first evidence for a differential pattern of synaptic organization of thalamostriatal glutamatergic inputs to the patch and matrix compartments. Furthermore, they demonstrate that the PF is the sole source of significant axodendritic thalamic inputs to striatal projection neurons. These observations pave the way for understanding differential regulatory mechanisms of striatal outflow from the patch and matrix compartments by thalamostriatal afferents.

Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia

All eukaryotic cells utilize oxidative phosphorylation to maintain their high-energy phosphate stores. Mitochondrial oxygen consumption is required for ATP generation, and cell survival is threatened when cells are deprived of O(2). Consequently, all cells have the ability to sense O(2), and to activate adaptive processes that will enhance the likelihood of survival in anticipation that oxygen availability might become limiting. Mitochondria have long been considered a likely site of oxygen sensing, and we propose that the electron transport chain acts as an O(2) sensor by releasing reactive oxygen species (ROS) in response to hypoxia. The ROS released during hypoxia act as signalling agents that trigger diverse functional responses, including activation of gene expression through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-alpha. The primary site of ROS production during hypoxia appears to be complex III. The paradoxical increase in ROS production during hypoxia may be explained by an effect of O(2) within the mitochondrial inner membrane on: (a) the lifetime of the ubisemiquinone radical in complex III; (b) the relative release of mitochondrial ROS towards the matrix compartment versus the intermembrane space; or (c) the ability of O(2) to access the ubisemiquinone radical in complex III. In summary, the process of oxygen sensing is of fundamental importance in biology. An ability to control the oxygen sensing mechanism in cells, potentially using small molecules that do not disrupt oxygen consumption, would open valuable therapeutic avenues that could have a profound impact on a diverse range of diseases.

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

Radiation therapy encompassing an active epiphysis can negatively impact the potential for bone growth by disrupting cell-cycle progression and accelerating apoptosis and terminal differentiation in physeal chondrocytes. Despite functional derangement following radiation exposure, the irradiated growth plate retains a capacity for regeneration and recovery of growth. The purpose of this study was to characterize the initial sequence of events leading to functional growth recovery in irradiated weanling rat growth plates. We hypothesized that growth in an irradiated epiphysis would be partially restored due to the expansion of chondrocytic clones. Stereological histomorphometry was used to compare chondrocytic cell and matrix turnover between the first and second week following irradiation, and to determine the relative contribution of each of the cellular and extracellular matrix (ECM) compartments to growth. We found that restoration of growth in the irradiated limb was strongly associated with the proliferative activity and production of ECM by these chondrocytic clones, as they expand in average volume, but not in numerical density. We conclude that chondrocytes forming expansive clones and exhibiting increased mitotic and matrix synthesis activity initiate the early restoration of function in the irradiated growth plate, and would be a logical target for strategies to restore full growth potential.

A Computational Model Integrating Electrophysiology, Contraction, and Mitochondrial Bioenergetics in the Ventricular Myocyte

An intricate network of reactions is involved in matching energy supply with demand in the heart. This complexity arises because energy production both modulates and is modulated by the electrophysiological and contractile activity of the cardiac myocyte. Here, we present an integrated mathematical model of the cardiac cell that links excitation-contraction coupling with mitochondrial energy generation. The dynamics of the model are described by a system of 50 ordinary differential equations. The formulation explicitly incorporates cytoplasmic ATP-consuming processes associated with force generation and ion transport, as well as the creatine kinase reaction. Changes in the electrical and contractile activity of the myocyte are coupled to mitochondrial energetics through the ATP, Ca 2+, and Na + concentrations in the myoplasmic and mitochondrial matrix compartments. The pseudo steady-state relationship between force and oxygen consumption at various stimulus frequencies and external Ca 2+ concentrations is reproduced in both model simulations and direct experiments in cardiac trabeculae under normoxic conditions, recapitulating the linearity between cardiac work and respiration in the heart. Importantly, the model can also reproduce the rapid time-dependent changes in mitochondrial NADH and Ca 2+ in response to abrupt changes in workload. The steady-state and dynamic responses of the model were conferred by ADP-dependent stimulation of mitochondrial oxidative phosphorylation and Ca 2+-dependent regulation of Krebs cycle dehydrogenases, illustrating how the model can be used as a tool for investigating mechanisms underlying metabolic control in the heart.

Bone‐specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma protein

The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGFbeta1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair.

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met(35), the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single A to G [corrected] mutation converts Grx2 into a typical protein of the mitochondrial matrix. The "A" denotes adenine, rather than alanine, and the "G" refers to guanine, not glycine [corrected]

Elevated level of ambient glucose stimulates the synthesis of high-molecular-weight hyaluronic acid by human mesangial cells. The involvement of transforming growth factor beta1 and its activation by thrombospondin-1.

The dysregulation of the metabolism of glycosaminoglycan and protein components of extracellular matrix (ECM) is a typical feature of diabetic complications. High glucose-induced enrichment of ECM with hyaluronan (HA) not only affects tissue structural integrity, but influences cell metabolic response due to the variety of effects depending on the HA polymer molecular weight. TSP-1-dependent activation of TGFbeta1 axis is known to mediate numerous matrix disorders in diabetes, but its role concerning HA has not been studied so far. In this work we demonstrated that 30 mM D-glucose increased the incorporation of [(3)H]glucosamine in high-molecular-weight (> 2000 kDa) HA of medium and matrix compartments of human mesangial cultures. Simultaneously, the synthesis of HA with lower molecular weight and HA degradation were not altered. The cause of the increased high-molecular-weight HA synthesis consisted in the up-regulation of hyaluronan synthase (HAS) 2 mRNA without alterations of the expression of HAS3, which generates HA of lower molecular weight. D-Glucose at 30 mM also stimulated the production of transforming growth factor beta1 (TGFbeta1), the excessive activation of which was determined by the up-regulation of thrombospondin-1 (TSP-1). The blockage of TGFbeta1 action either by neutralizing anti-TGFbeta1 antibodies or by quenching the TGFbeta1 activation (with TSP-1-derived synthetic GGWSHW peptide) abolished the effect of high glucose on HAS2 mRNA expression and normalized the synthesis of HA. Exogenous human TGFbeta1 had the same effect on HAS2 expression and HA synthesis as high glucose treatment. Therefore, we supposed that TSP-1-dependent TGFbeta1 activation is involved in the observed high glucose effect on HA metabolism. Since high-molecular-weight HA polymers, unlike middle- and low-molecular weight HA oligosaccharides, are known to possess anti-inflammatory and anti-fibrotic functions, we suppose that the enrichment of mesangial matrix with high-molecular-weight HA may represent an endogenous mechanism to limit renal injury in diabetes.

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

Les agents de régénération (ou RGTAs) : une nouvelle approche thérapeutique

RGTAs, or ReGeneraTing Agents constitute a new class of medicinal substance that enhance both speed and quality of tissue healing and leading in some case to a real tissue regenerating process. RGTAs consist of chemically engineered polymers adapted to interact with and protect against proteolytic degradation of cellular signaling proteins known as growth factors, cytokines, interleukins, colony stimulating factors, chemokines, neurotrophic factors etc. Indeed almost all these proteins of cellular communication are naturally stored in the extra cellular matrix interacting specifically with the heparan sulfates or HS. After tissue injury of any cause, cells die liberating glycanases and proteases inducing first HS degradation then liberation of the cytokines which in turn are susceptible to degradation as they are no longer protected. By replacing the natural HS, RGTAs will protect cytokines from proteolyses as they are liberated from the matrix compartment matter in the wound. This spatio-temporal selective protection of cytokines results in a preservation of the natural endogenous signaling of a tissue and is reflected by spectacular tissue regeneration or by a very greatly improved tissue repair. These observations indicate that mammals have an unexpected ability to regenerate and that RGTA helps to reveal this capacity. The aim of OTR3 is to develop RGTA into a drug to treat specific tissue lesions.

Regulation of psychostimulant-induced preprodynorphin, c-fos and zif/268 messenger RNA expression in the rat dorsal striatum by mu opioid receptor blockade

Several studies have shown that psychostimulants can induce differential immediate early gene and neuropeptide expression in the patch versus matrix compartments of dorsal striatum. The patch compartment contains a high density of mu opioid receptors and activation of these receptors may contribute to psychostimulant-induced gene expression in the patch versus matrix compartments of dorsal striatum. However, the contribution of mu opioid receptor activation to psychostimulant-induced changes in gene expression in the patch compartment of dorsal striatum has not been examined. The current study examined the role of mu opioid receptors in psychostimulant induction of preprodynorphin, c-fos and zif/268 messenger RNA expression in the patch versus matrix compartments of dorsal striatum. Male Sprague-Dawley rats were treated with the mu opioid receptor antagonist, clocinnamox (1 mg/kg, s.c.), 24 h prior to treatment with cocaine (30 mg/kg, i.p.) or methamphetamine (15 mg/kg, s.c.) and sacrificed 45 min or 3 h later. Mu opioid receptor antagonism blocked psychostimulant-induced preprodynorphin messenger RNA expression only in the rostral patch compartment, whereas psychostimulant-induced zif/268 messenger RNA expression in the patch and matrix compartments was attenuated throughout the dorsal striatum. Clocinnamox pretreatment had no effect on stimulant-induced increases in c-fos expression. These data suggest that mu opioid receptor activation plays a specific role in psychostimulant-induced preprodynorphin messenger RNA expression in the rostral patch compartment and zif/268 messenger RNA expression throughout dorsal striatum.

Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy

Differential regulation of metzincins in experimental chronic renal allograft rejection: Potential markers and novel therapeutic targets

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

A novel “sandwich” assay for quantifying chemo-regulated cell migration within 3-dimensional matrices: Wound healing cytokines exhibit distinct motogenic activities compared to the transmembrane assay

The extracellular matrix profoundly affects cellular response to soluble motogens. In view of this critical aspect of matrix functionality, we have developed a novel assay to quantify chemo-regulated cell migration within biologically relevant 3-dimensional matrices. In this "sandwich" assay, target cells are plated at the interface between an upper and lower matrix compartment, either in the presence of an isotropic (uniform) or anisotropic (gradient) spatial distribution of test motogen. Cell migration in response to the different conditions is ascertained by quantifying their subsequent disposition within the upper and lower matrix compartments. The objective of this study has been to compare the motogenic activities of platelet-derived growth factor (PDGF-AB) and transforming growth factor-beta isoforms (TGF-beta1, -beta2 and -beta3) in the sandwich assay and the commonly employed transmembrane assay. As previously reported, dermal fibroblasts exhibited a motogenic response to isotropic and anisotropic distributions of all tested cytokines in the transmembrane assay. In contrast, only PDGF-AB and TGF-beta3 were active in the sandwich assay, each eliciting directionally unbiased (symmetrical) migration into the upper and lower type I collagen matrices in response to an isotropic cytokine distribution and a directionally biased response to an anisotropic distribution. TGF-beta1 and -beta2 were completely devoid of motogenic activity. These results are consistent with the reported differential bioactivities of PDGF and TGF-beta3 compared to TGF-beta1 and -beta2 in animal models of wound healing and suggest that the sandwich assay provides a means of obtaining physiologically relevant data regarding chemo-regulated cell migration.

Submitochondrial localization of the mitochondrial isoform of folylpolyglutamate synthetase in CCRF-CEM human T-lymphoblastic leukemia cells

Earlier studies from this laboratory showed that human folylpolyglutamate synthetase (FPGS) exists as cytosolic and mitochondrial (mFPGS) isoforms. Localization of mFPGS within mitochondria may help elucidate how the enzyme functions to maintain the mitochondrial folate pool. A human T-lymphoblastic leukemia CCRF-CEM cell lysate was fractionated by differential centrifugation into cytosolic and mitochondrial fractions. Activity assays for cytosol-and mitochondria-specific enzymes verified the purity and integrity of the fractions. Mitochondria were subfractionated with increasing concentrations of digitonin to successively extract the four submitochondrial compartments. Western analyses of the fractions using protein markers specific for each compartment suggest that mFPGS is distributed in the matrix and/or inner membrane compartments. Further support for an interaction of mFPGS with the inner mitochondrial membrane is provided by localization of about half of the mFPGS in the mitochondrial membrane fraction obtained by freeze–thaw of intact mitochondria; the remaining mFPGS is located in the soluble fraction. Resistance of about half of the mFPGS in whole mitochondria to alkaline carbonate extraction suggests that its interaction with the inner membrane is more similar to an integral, than a peripheral, membrane protein. The data suggest that human mFPGS is at least in part strongly associated with the inner mitochondrial membrane.

ATP production in Chlamydomonas reinhardtii flagella by glycolytic enzymes.

Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies reported previously in the cpc1 strain. We conclude that in situ ATP synthesis throughout the flagellar compartment is essential for normal flagellar motility.