Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa.

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections.

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test-point lattices on cell profiles, design-based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test-line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds microm(-2) or membranes (golds microm(-1)), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi-squared analysis to test if the observed distribution differs significantly from random and, if it is non-random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.

Electron microscopy applications for quantitative cellular microbiology.

Recently, there has been a surge of interest in analysing intracellular trafficking of pathogenic microorganisms. This field relies heavily on a wide range of approaches developed by cell biologists. However, one well-established approach that seems to be underused in cellular microbiology is electron microscopy (EM). In this article, we emphasize the power of state-of-the-art EM approaches, especially when used in conjunction with the quantitative approaches provided by stereology. Together, this combination of techniques can be exploited to pursue kinetic analyses of the intracellular pathways followed by microorganisms. In addition, it will provide a more detailed description of the antigenic composition of relevant structures than is possible by light microscopy (LM). Here, we will explain how EM, applied in an integrated approach with LM, has distinct advantages and can provide unique insights into the cellular fate of microorganisms.

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network.

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.

Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi.

We have identified a novel protein of predicted molecular mass 40 kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain.

The manganese cation disrupts membrane dynamics along the secretory pathway.

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Okadaic acid induces selective arrest of protein transport in the rough endoplasmic reticulum and prevents export into COPII-coated structures.

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15 degrees C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.

Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells.

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.

Carbonic anhydrase isoenzymes I, II, III, and IV are present in human esophageal epithelium.

Carbonic anhydrase (CA) isoenzymes have been widely studied in the gastrointestinal tract, where they mediate membrane transport events and pH regulation. However, the esophagus has generally received scant attention. In an immunohistochemical study confirmed by Western blotting, we have detected for CA isoenzymes (CAI, II, III, and IV) in the epithelium of human esophagus. Isoenzymes I, III, and sometimes IV (< 10%) were present in the cytoplasm of basal cells and II and IV in the cytoplasm and cell surface membranes, respectively, of suprabasal cells (prickle cells). The localization of CAIV to the plasma membranes was confirmed by electron microscopic immunocytochemistry. CA was effectively divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal). Carbonic anhydrase in esophageal epithelial cells may have several functions: elimination of CO2 and metabolites, participation in membrane transport events during active cell growth, and pH regulation as a protective mechanism against acidic gastric reflux.

Low extracellular pH induces activation of ERK 2, JNK, and p38 in A431 and Swiss 3T3 cells.

The mechanism by which mammalian cells respond to low environmental pH is unclear. A wide range of environmental stresses are known to induce activation of MAP kinases ERK 2, JNK and p38 and recent work has shown that low pH can activate the p38 homologue in yeast HOG1. In this study we show that ERK2 MAP kinase is activated in human A431 cells exposed to low pH media. Activation is sustained throughout low pH treatment, is reversible, and occurs maximally at pH 4 or 5. Stimulation is not accompanied by tyrosine phosphorylation of the EGF receptor or Raf-1 activation, indicating that acid conditions act via pathways independendent of those required for EGF mediated MAPK stimulation. The MAP kinase homologue JNK and MAPKAP kinase-2 reactivating kinase (p38) were also activated in A431 cells by low pH and so low pH induces parallel activation of multiple MAP kinase pathways. Strong activation of p42, and p44 ERKs as well as p38 and JNK was also found in mouse Swiss 3T3 cells treated at pH 5. These results indicate that MAP kinases may be important markers of the acid induced cellular stress that occurs in human disease.

Role of translocation in the activation and function of protein kinase B.

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKBalpha was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis demonstrated that m/p-PKBalpha activity was due to phosphorylation on Thr308 and Ser473, that are normally induced on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) did not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha was not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha was translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation required the PH domain and was sensitive to wortmannin. Our results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detached from the membrane and translocated to the nucleus.

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein.

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.

The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum.

The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expressed in the basolateral membrane (BLM) of the human intestine. BBM and BLM were isolated by fractionation of human jejunum. BBM were enriched with alkaline phosphatase activity by over 9-fold relative to a crude jejunal homogenate and contained immunoreactive sucrase-isomaltase and GLUT5 proteins. By contrast the BBM fraction was substantially depleted of immunoreactive a1 subunits of the Na,K-ATPase and GLUT2 glucose transporters which were abundantly present in the BLM fraction. This BLM fraction was enriched by over 11-fold in potassium-stimulated phosphatase activity relative to the crude homogenate; BLM also reacted to immunological probes for GLUT5 but showed no observable reactivity with antibodies directed against sucrase-isomaltase. Quantitative immunoblotting revealed that the BBM and BLM contained near equal amounts of GLUT5 per mg of membrane protein. Immunogold localization of GLUT5 on ultrathin sections of human jejunum showed that GLUT5 was present in both apical BBM and BLM. This gold labelling was absent when antiserum was pre-incubated with the antigenic peptide corresponding to a specific C-terminal sequence of human GLUT5. Quantitative analyses of the number of gold particles per unit length of BBM and BLM indicated that the mean density of gold labelling was marginally greater in the BBM (0.399 gold particles/micrometer) than in the BLM (0.293 gold particle/micrometer). The localization of GLUT5 in the BLM of the human jejunum may suggest that it specifically participates in the transfer of fructose across the basal membrane of the enterocyte.

Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells.

The peptide/class II major histocompatibility complex (MHC) complexes recognized by CD4+ T cells have been characterized at the structural and biochemical levels and studies on the transport and maturation of class II MHC indicate that specialized sites may be involved in peptide acquisition. Here we report the characterization of the compartments involved in antigen processing and class II MHC loading relative to distinct functional domains of the endocytic pathway in antigen-specific human B lymphocytes. Peroxidase-mediated crosslinking analysis in intact cells demonstrates that peptide loading of class II MHC takes place in a compartment accessible to membrane immunoglobulin but not to transferrin receptors, although processing may be initiated within the latter domain. The density of membrane vesicles carrying newly assembled class II MHC complexes was distinct from early and late endosomes and dense lysosomes. Endocytosed antigen-gold complexes enter a class II MHC-rich compartment morphologically very similar to that described previously and within the time frame of biochemically detectable peptide loading.

A 40-kDa myelin basic protein kinase, distinct from erk1 and erk2, is activated in mitotic HeLa cells.

Mitotic HeLa cells showed an increased phosphorylation activity towards myelin basic protein compared to cells in G1 or S phases. Further investigation using renaturation gels revealed that, in mitotic cell lysates, a protein with an apparent molecular mass of around 40 kDa phosphorylates myelin basic protein. This kinase is active early in mitosis, but is then downregulated concomitantly with p34cdc2 kinase as mitosis proceeds, its activity decreasing to basal levels by early G1. The molecular mass of the kinase suggested that it might be one of the human homologues of rat erk1 or erk2. However, antibodies raised against C-terminal sequences of erk1 and erk2 failed to immunoprecipitate renaturable kinase activity from mitotic lysates. In addition, in immunoblots erk1 and erk2 failed to show the well established changes in electrophoretic migration that are consequences of their activation. These data indicate that these two mitogen-activated protein (MAP) kinases are not stimulated during HeLa cell mitosis and indicate that the 40-kDa kinase is either a new member of the MAP kinase family or it is a novel mitotic kinase that has not yet been described.

Demonstration of pulmonary vascular perfusion by electron and light microscopy.

To estimate the fraction of dense pulmonary capillary network that is perfused under physiological conditions, we developed a new method for the demonstration of in vivo capillary perfusion by light and electron microscopy. Blood plasma was labeled by 8-nm colloidal gold particles coated with rabbit serum albumin. In anesthetized rabbits, 4-5 ml of this tracer were injected into the right atrium. Two and 15 min later, the circulation was interrupted by a snare around the heart, and the lung was fixed by instillation with glutaraldehyde. Gold particles were found in the plasma space of alveolar capillaries as well as in other organs. A random sample of thin sections studied by electron microscopy revealed that the entire capillary bed of the lung was perfused at least with plasma within 2 min after tracer infusion. Light microscopy of silver-enhanced sections showed areas with different staining intensities but no obviously unperfused capillaries. The concept of capillary recruitment, which would require a significant fraction of capillaries unperfused at rest, may have to be reassessed to consider time factors as well as the two-phase nature of blood; red blood cells and plasma may take different paths.

The membrane spanning domain of beta-1,4-galactosyltransferase specifies trans Golgi localization.

Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.