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PubMed1999

Compartmentalization of interphase chromosomes observed in simulation and experiment.

Human interphase chromosomes were simulated as a flexible fiber with excluded volume interaction, which represents the chromatin fiber of each chromosome. For the higher-order structures, we assumed a folding into 120 kb loops and an arrangement of these loops into rosette-like subcompartments. Chromosomes consist of subcompartments connected by small fragments of chromatin. Number and size of subcompartments correspond with chromosome bands in early prophase. We observed essentially separated chromosome arms in both our model calculations and confocal laser scanning microscopy, and measured the same overlap in simulation and experiment. Overlap, number and size of chromosome 15 subcompartments of our model chromosomes agree with subchromosomal foci composed of either early or late replicating chromatin, which were observed at all stages of the cell cycle and possibly provide a functionally relevant unit of chromosome territory compartmentalization. Computed distances of chromosome specific markers both on Mb and 10-100 Mb scale agree with fluorescent in situ hybridization measurements under different preparation conditions.

Cell Cycle↗
PubMed1997

Hepatic foci in rats after diethylnitrosamine initiation and 2,3,7,8-tetrachlorodibenzo-p-dioxin promotion: evaluation of a quantitative two-cell model and of CYP 1A1/1A2 as a dosimeter.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent hepatic tumor promoter in female rats. We used a quantitative, stochastic initiation-promotion model based on R. B. Conolly and J. S. Kimbell (Toxicol. Appl. Pharmacol. 124, 284-295, 1994) to analyze initiation-promotion results from a previously published study (H. C. Pitot et al., Carcinogenesis 8, 1491-1499, 1987) within the context of a negative selection model of tumor promotion. In this model, two types of initiated cells (called A and B cells) are produced by DEN initiation. Visually excellent correspondence between model predictions and data (i.e., foci/cm3 liver and percentage of liver occupied by foci) are obtained when TCDD is described as having dose-responsive effects on division and death (apoptotic) rates of these two cell types. For A cells, both the division and the death rates increase while the difference between division and apoptotic rates decreases. For B cells, the difference between division and apoptotic rates increases, primarily due to a decrease in the apoptotic rate. We also linked these alterations in cell kinetics to a pharmacokinetic model for TCDD incorporating a five subcompartment model of the liver acinus with induction of CYP1A1 and 1A2 genes in the subcompartments. Alterations in A cell kinetics correlate with effects of TCDD in the region most sensitive to induction (subcompartment 5-centrilobular region); B cell dynamics correlate with induction in subcompartments 3-5 (centrilobular and mid-zonal regions). In summary, these modeling exercises show that (1) the two-cell model, without presuming effects of TCDD on the mutation rate of normal hepatocytes, reproduces the data of Pitot et al. (1987) and (2) induction of CYP1A1/1A2 in different regions of the hepatic acinus can be used as a general correlate of these presumed changes in cell growth kinetics.

Animals↗
PubMed1990

Time course of the quantitative changes in the autophagic-lysosomal and secretory granule compartments of murine liver cells under the influence of vinblastine.

The dynamics of the transient expansion of the autophagic-lysosomal (ALC) and secretory granule (SGC) compartments in mouse liver cells were monitored by electron microscopic morphometry after a single injection of 10 mg/kg b.w. vinblastine sulfate (VBL). Initially (first phase) the cytoplasmic volume fractions of the total ALC and its subcompartments, as well as of the SGC increased by an order of magnitude and peaked at the second h. In the second phase, all the aforementioned compartments regressed gradually, approaching their normal size between 12 and 36 h after VBL injection. Analysis of the dynamic changes in fractional volumes of subcompartments of the ALC showed that early autophagic vacuoles (AV1) were the first to enlarge. Advanced AVs (AV2) reacted 30 min later and a further 30 min time lag was required before late autolysosomes, appearing as dense bodies (DB), started to expand. We regard these data as kinetic proof that the bodies of the later reacting subcompartments developed from the earlier reacting ones. The time lag between the expansion of AV1 and AV2 subcompartments may be explained by a period of retardation of conversion of nascent autophagosomes (AV1) to autolysosomes (AV2) which is known to occur normally by fusion of AV1 with enzyme-carrying lysosomes. However, transformation of AV1 to AV2 and later to DB resumed after the respective time lags. Moreover, our quantitative data lend support to the view that segregation of cytoplasmic portions into newly-formed autophagosomes was stimulated by VBL, at least in the first 2 h of treatment. The expansion of ALC accelerated during this period and led to an obvious overload of the lysosomal apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

A comparison of abdominal subcutaneous adipose tissue pattern in obese and lean HIV-infected women.

Cells from the superficial and deep subcompartments of the abdominal subcutaneous adipose tissue (SAT) compartment have distinct metabolic activities in vitro. The effect of differing energy balance on the relative in vivo sizes of these subcompartments has not been reported. We retrospectively investigated the effects of obesity and leanness on the relative amounts of superficial and deep SAT in the bulky posterior abdominal adipose tissue in HIV(+) women. We studied the baseline results of MRI scans in 32 obese and 28 lean HIV-infected women. We also compared the change in response to specific interventions. Abdominal MRI slices were obtained at the L4-L5 and L2-L3 intervertebral spaces and were divided into anterior and posterior halves. The posterior portions were further subdivided into deep (PDSAT) and superficial layers (PSSAT) based on tissue planes visible on the MRI. Fat areas in adjacent landmark levels at the trochanter and anterior superior iliac spine were also obtained. PDSAT was larger at L4-L5 than at L2-L3 in both the lean and obese groups. PDSAT was larger than PSSAT at L4-L5 in obese women, and there was preferential loss of PDSAT in obese women who completed a 12-wk energy-deficit diet and exercise program. The contents of PDSAT and PSSAT did not differ in the lean group, and proportional increases in both SAT subcompartments were noted in response to weight gain. In summary, obesity is associated with a preferential increase in PDSAT and greater loss in PDSAT after weight loss. This study defines distinct metabolism responses in fat subcompartments.

Abdomen↗
PubMed2004

Site-specific metabolism of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques.

Studies in rodents have demonstrated the importance of cytochrome P450 monooxygenases in generating reactive metabolites that produce Clara cell injury. Pulmonary P450 activities in rodents are much higher than those in primates, raising the issue of relevance of rodent data to primates. Few studies on P450-catalyzed activation of cytotoxicants in subcompartments of primate lung have been reported. Accordingly, infant monkey airway subcompartments, including trachea, proximal, midlevel, distal airways, and parenchyma, were incubated with naphthalene or 1-nitronaphthalene to define metabolism at both high (500 microM) and low (50 microM) substrate concentrations. There was a relatively even distribution of metabolizing activities for naphthalene across subcompartments, but at high concentrations of 1-nitronaphthalene, lower airways (midlevel airway through parenchyma) showed higher bioactivation than upper airways. Dihydrodiol was the predominant water-soluble metabolite of naphthalene generated by all subcompartments, whereas covalently bound metabolites accounted for the greatest percentage of 1-nitronaphthalene metabolites, especially in lower airways. As anticipated, the amounts of metabolite covalently bound as a percentage of total metabolite formed increased dramatically with the 10-fold increase in substrate concentration. With both substrates, the formation of water-soluble metabolites was approximately 100 times less than observed previously in rodents. We conclude that 1) there are significant quantitative differences between rhesus and rodents in substrate bioactivation; 2) the distribution of metabolizing activities for naphthalene but not 1-nitronaphthalene is significantly different for rodents and primates; and 3) a very high percentage of the metabolites generated, particularly for 1-nitronaphthalene, is bound covalently to cellular proteins.

Animals↗
PubMed1988

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells.

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.

Animals↗
PubMed1999

Morphology of feedback neurons in the mushroom body of the honeybee, Apis mellifera.

The anatomy of gamma-aminobutyric acid (GABA)-immunoreactive, recurrent feedback neurons in the mushroom body (MB) of the honeybee, Apis mellifera, was investigated by using intraneuropilar injections of cobalt ions and light microscopic techniques. Each MB contains approximately 110 GABA-immunoreactive neurons, and approximately 50% of them are feedback neurons, i.e., they connect the MB output regions--the alpha-lobe, beta-lobe, and pedunculus--with its input regions--the calyces. Their somata are located in the lateral protocerebral lobe, and their primary neurites project medially and bifurcate near the alpha-lobe. In the alpha-lobe feedback neurons form narrow banded, horizontal arborizations in the dorsal and median alpha-lobe; each cell innervates a certain alpha-lobe layer. The neurons form additional branches in the pedunculus and the beta-lobe. All calycal subcompartments--the lip, collar, and basal ring--are innervated by feedback neurons. However, individual feedback neurons innervate exclusively a certain subcompartment in both the median and lateral calyx. Due to the arrangement of intrinsic Kenyon cells, each calycal subcompartment is connected to its specific, corresponding layer in the alpha-lobe. Feedback neurons interconnect the alpha-lobe and the calyces in either a corresponding or a noncorresponding fashion. With respect to their branching pattern in the alpha-lobe, the basal ring and the collar neuropil receive input from feedback neurons innervating the corresponding dorsal and median alpha-lobe layers. By contrast, the lip region, which receives olfactory antennal input, is innervated by feedback neurons with arborizations in a noncorresponding dorsal alpha-lobe layer.

Animals↗
PubMed1984

Serotonin-immunoreactive neurons in the brain of the honeybee.

The distribution of serotonin-immunoreactive neurons in the brain of the worker honey bee Apis mellifera was studied by means of immunocytochemical staining by using a well-characterized antibody to serotonin (5-HT). About 75 immunoreactive perikarya are grouped into clusters in the optic lobe and in the median and dorsal protocerebrum. Immunoreactive fibers were resolved in all areas of the brain. The optic lobe shows restricted layers of 5-HT-immunoreactive fibers in the lamina and medulla organized perpendicular to the retinotopic elements. Immunoreactive fibers in the lobula represent invasions of protocerebral giant wide-field neurons. The nonglomerular neuropil of the brain exhibits a meshwork of immunoreactive fibres invading glomerular neuropil of the mushroom bodies, central body complex, and antennal lobes. Mushroom body stalks and lobes contain immunoreactive fibers arranged perpendicular to the Kenyon cell fibers and matching subcompartments of these corpora pedunculata areas. The calyces are devoid of immunofluorescence. Serotonin-positive fibres in the central body complex are arranged in its subcompartments. No 5-HT immunoreactivity was found in the pons. Antennal glomeruli contain immunoreactive fibers restricted around the margin of the glomeruli. The selective mapping of 5-HT-immunoreactive neurons complements studies on the distribution of monoamine-containing neurons in the bee brain. Serotonin- and catecholamine-containing neurons often occur together in the same brain areas and subcompartments. The immunohistochemical approach in chemoneuroanatomy gives new evidence for a more complicated architecture of the brain than could be deduced from the classical neuroanatomical studies.

Animals↗
PubMed1990

Flow cytometric analysis of G1- and G2/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements.

Several subcompartments of the cell cycle in addition to the G1-, S-, and G2-phases usually observed were identified by simultaneous flow cytometric measurements of ethidium bromide fluorescence and side scatter intensity of cell nuclei. Metaphase cells and very early G1-phase cells (G1A) with low side scatter intensities were discriminated from interphase cells with high side scatter intensities. The reason for the various side scatter intensities was found to be the different structure of metaphase cells and early G1-phase cells due to chromatin condensation as shown by sorting of the respective cell nuclei. The G1A-phase could further be subdivided into two compartments with very low side scatter (G1A1) and intermediate side scatter (G1A2) intensities. Using partially synchronized cells the duration of these subcompartments of the G1-phase could be estimated. The durations of G1A1- and G1A2-phases were found to be about 10 min and 20 min, respectively, compared to the total duration of the G1-phase of about 3 h. Additional flow cytometric measurements of side scatter intensities of cell nuclei provide therefore further information on subcompartments of the G1- and G2/M-phases.

Animals↗
PubMed1991

Cytochemical characteristics of the Golgi apparatus.

Lectinocytochemistry provides a useful tool for localizing subcompartments of the complex reticular apparatus of Golgi. The technique is based on interactions of lectins with glycoconjugates present in the limiting membranes and luminal spaces of Golgi elements. Application of a series of lectins of different sugar specificities permits a differentiation between Golgi subcompartments containing glycoconjugates with different oligosaccharide side chains. These may be a) differnet glycoconjugates or b) glycoconjugates at different stages during synthesis or repair of their glycans. The lectinocytochemical studies with mannose-, glucose-, N-acetyl-glucosamine-, N-acetyl-galactosamine-, galactose-, fucose-, and sialic acid-recognizing lectins revealed predominating patterns that labeled distinct, i.e., cis, medial, trans, and transmost, regions of the Golgi apparatus. A further refinement could be achieved by differential lectin-inhibition that enables a dissection of lectin binding reactions on the basis of their binding affinities. High-affinity binding reactions showed that subcompartments are not necessarily confined to one single Golgi subregion and may change their position from one to another subregion. Some of the patterns observed may be interpreted in relation to certain steps during synthesis and modifications of glycans.

Animals↗
PubMed1994

Dose-dependent tolerance to ozone. IV. Site-specific elevation in antioxidant enzymes in the lungs of rats exposed for 90 days or 20 months.

Ozone-induced lung injury in rats is focal, with the primary target sites being the distal trachea and the central acinus. In both area, ozone causes cellular injury and necrosis after short-term exposures, but the areas become tolerant to further injury after long-term exposure. To investigate the role of antioxidant enzymes in the resistance of the lung to injury from long-term ozone exposure, we measured activities of three antioxidant enzymes in airway samples microdissected from specific sites within the lung: distal trachea, lobar bronchi, major daughter axial bronchi, minor daughter bronchi, distal bronchiole, and parenchyma. Fischer 344 rats were exposed to 0, 0.5, and 1 ppm ozone 6 hr/day, 5 days/week for 20 months, or to 0, 0.12, and 1 ppm for 90 days. Glutathione transferase, glutathione peroxidase, and superoxide dismutase activities were measured at the end of the exposure periods. Data were normalized for DNA content (Units/mg DNA). For both the 90-day and 20-month exposures, the activities of all three enzymes were significantly elevated in a concentration-dependent fashion in the distal bronchioles. Compared to controls, animals exposed to 1.0 ppm ozone had superoxide dismutase activities 1.6x (90 days) and 2x (20 months) greater; glutathione peroxidase had activities 1.4x (90 days) and 1.6x (20 months) greater; and glutathione S-transferase had activities 1.5x (90 days and 20 months) greater. In animals exposed for 90 days, superoxide dismutase activity was lower in major daughter bronchi and greater in minor daughter bronchi and glutathione peroxidase activity was lower in major daughter bronchi. After 20 months of exposure, superoxide dismutase activity was significantly elevated in a dose-dependent fashion in the distal trachea; glutathione peroxidase activity decreased in the major daughter bronchi and increased in the minor daughter bronchi; and glutathione S-transferase activity decreased in the major daughter bronchi. There were no changes in antioxidant enzyme levels in other subcompartments. Superoxide dismutase activity increased in a concentration-dependent fashion in the whole lung homogenate of animals exposed for 90 days, but no differences were detected in whole lung homogenates of any other exposure groups. We conclude that (1) antioxidant enzyme activities are altered on a site-specific basis in response to long-term exposure to ozone; (2) the antioxidant enzymes respond differently in different lung subcompartments; (3) activities determined for the whole lung do not reflect changes in subcompartments with variable susceptibility to injury; and (4) changes in antioxidant enzyme activities are concentration-dependent and altered by length of exposure.

Animals↗
PubMed1997

A morphological anomaly of the dorsal lateral geniculate nucleus in Macaca fascicularis.

In the present report we describe a morphological anomaly of the thalamus. In three macaque monkeys (Macaca fascicularis), we observed up to five finger-like protrusions that emanated from the posterior pole of the dorsal lateral geniculate nucleus (LGN) and extended posteriorly between the lateral pulvinar and reticular nucleus of the thalamus. These anomalous fingers measured up to 1.7 mm in length and contained dense accumulations of neurons and glia. The fingers received a direct retinal input from the contralateral eye indicating that they were part of the LGN rather than of other adjacent thalamic nuclei. In order to determine with which subcompartment(s) of the LGN the fingers were associated (parvocellular, magnocellular, or intercalated layers), we examined the immunochemical properties and size of neurons in the fingers and LGN subcompartments. We concluded that the fingers were not associated with the intercalated layers, since neurons in the fingers did not stain with an antibody to calbindin-D28k, whereas intercalated neurons stained intensely with this antibody. In addition, neurons located in the fingers were significantly smaller than those found in the magnocellular layers but were not significantly different in size from neurons in the parvocellular layers. We therefore consider that the fingers are an anomaly of the parvocellular subcompartment of the LGN. Interestingly, in two of the three cases with anomalous fingers, we also observed subsidiary parvocellular laminae, suggesting that these two anomalies were related. In five additional animals, however, we observed subsidiary parvocellular laminae without anomalous fingers. Thus, if there are common mechanisms underlying the development of both anomalous fingers and subsidiary layers, our data indicate that they do not always result in the concomitant expression of both anomalies.

Animals↗
PubMed2001

A hierarchy of the functional organization for color, form and disparity in primate visual area V2.

By combining optical imaging, single unit electrophysiology and cytochrome oxidase (CO) histology, we sought to reveal in greater detail the functional organization within the CO stripes of visual area V2 of primates. To visualize the disparity selective regions of V2, the imaging of binocular interaction was employed. These imaging maps guided single unit penetrations that then revealed a columnar organization for disparity. Our studies also showed a pattern of intermixing between the color and disparity pathways of V2, including the existence of single cells tuned for both color and disparity. While previous studies have suggested that the CO stripes of V2 constitute the fundamental organizational unit within V2, our results show a further level of organization consisting of functionally distinct subcompartments, 0.7-1.5 mm in diameter, within individual stripes. These subcompartments, which are not clearly revealed by CO histochemistry, lie within each of the thin, pale, and thick CO dense stripes in V2 and are specific for aspects of color, orientation and retinal disparity, respectively. The present results favor an architectural view of V2, not unlike that of V1, as a collection of functionally distinct subcompartments or modules situated within each of the V2 stripes. These modules also support the notion that for each cortical area (e.g. V1, V2, V4), there exists a stereotyped cortical module with a geometry that is characteristic for each area. These modules exist as a middle tier in a hierarchy of functional organization within V2.

Animals↗
PubMed1998

Dynamics of the interphase mammalian Golgi complex as revealed through drugs producing reversible Golgi disassembly.

We focus on research aimed at understanding normal Golgi complex dynamics through the use of nocodazole and other drugs which cause Golgi disassembly. In vivo nocodazole binds to tubulin, produces microtubule depolymerization, and subsequent fragmentation of the Golgi complex. These processes may be traced in living cells through the application of fluorescent green protein (GFP) conjugates. The cycling of individual Golgi proteins through the endoplasmic reticulum (ER) may be probed in vivo through the use of an organelle-specific molecular trap. One such molecular trap is protein unfolding. Golgi proteins conjugated with a domain temperature sensitive in protein folding exhibit temperature-sensitive folding properties and if misfolded during protein cycling from the Golgi become trapped in the ER. The properties of individual Golgi complex subcompartments may be characterized through antibodies to multiple subcompartment-specific proteins within the same cell line. Because of the limited availability of antibodies, normally distributed epitope tagged proteins are employed to give multiple subcompartment-specific Golgi complex markers. From experiments employing these tools, new models suggesting continuous cycling of Golgi proteins are emerging. Cycling of Golgi proteins through the ER can lead to assembly of the Golgi stack at or about ER exit sites. A major future challenge will be the characterization of the protein machineries involved in Golgi protein cycling and its regulation.

Animals↗
PubMed2003

Caveolin-1 regulates the functional localization of N-acetylglucosaminyltransferase III within the golgi apparatus.

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core beta-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density gradient ultracentrifugation. Caveolin-1 may modify the biosynthetic pathway of sugar chains via the regulation of the intra-Golgi subcompartment localization of this key glycosyltransferase.

Caveolin 1↗
PubMed1993

Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.

We report studies using an enhanced experimental system to investigate organization of nuclear pre-mRNA metabolism. It is based on the powerful genetic system and polytene nuclei of Drosophila. We observe (at steady state) movement of a specific pre-mRNA between its gene and the nuclear surface. This movement is isotropic, at rates consistent with diffusion and is restricted to a small nuclear subcompartment defined by exclusion from chromosome axes and the nucleolus. Bulk polyadenylated nuclear pre-mRNA precisely localizes in this same subcompartment indicating that most or all pre-mRNAs use the same route of intranuclear movement. In addition to association with nascent transcripts, snRNPs are coconcentrated with pre-mRNA in this subcompartment. In contrast to constitutive splices, at least one regulated splice occurs slowly and may undergo execution remotely from the site of pre-mRNA synthesis. Details of our results suggest that retention of incompletely spliced pre-mRNA is a function of the nuclear surface. We propose a simple model--based on channeled diffusion--for organization of intranuclear transport and metabolism of pre-mRNAs in polytene nuclei. We argue that this model can be generalized to all metazoan nuclei.

Animals↗
PubMed1998

Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins.

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.

Animals↗
PubMed2023

Mega-Enhancer Bodies Organize Neuronal Long Genes in the Cerebellum.

Dynamic regulation of gene expression plays a key role in establishing the diverse neuronal cell types in the brain. Recent findings in genome biology suggest that three-dimensional (3D) genome organization has important, but mechanistically poorly understood functions in gene transcription. Beyond local genomic interactions between promoters and enhancers, we find that cerebellar granule neurons undergoing differentiation in vivo exhibit striking increases in long-distance genomic interactions between transcriptionally active genomic loci, which are separated by tens of megabases within a chromosome or located on different chromosomes. Among these interactions, we identify a nuclear subcompartment enriched for near-megabase long enhancers and their associated neuronal long genes encoding synaptic or signaling proteins. Neuronal long genes are differentially recruited to this enhancer-dense subcompartment to help shape the transcriptional identities of granule neuron subtypes in the cerebellum. SPRITE analyses of higher-order genomic interactions, together with IGM-based 3D genome modeling and imaging approaches, reveal that the enhancer-dense subcompartment forms prominent nuclear structures, which we term mega-enhancer bodies. These novel nuclear bodies reside in the nuclear periphery, away from other transcriptionally active structures, including nuclear speckles located in the nuclear interior. Together, our findings define additional layers of higher-order 3D genome organization closely linked to neuronal maturation and identity in the brain.

Journal Article↗