Deconvolution Immunofluorescence Microscopy Research Articles

Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells.

Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10–18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

Common traffic routes for imported spermine and endosomal glypican-1-derived heparan sulfate in fibroblasts

Import of the polyamine spermine from the extracellular environment depends on the presence of cell surface heparan sulfate proteoglycans, such as glypican-1. This proteoglycan is internalized by endocytosis, releases its heparan sulfate chains in endosomes by a nitric oxide-, copper- and amyloid precursor protein-dependent mechanism, then penetrates the membrane and is transported to the nucleus and then to autophagosomes. This process is spontaneous or induced by ascorbate depending on the growth-state of the cell. Here, we have explored possible connections between the heparan sulfate traffic route and spermine uptake and delivery in wild-type and Tg2576 mouse fibroblasts. Cells were examined by deconvolution immunofluorescence microscopy. The antibodies used were specific for spermine, glypican-1-derived heparan sulfate, Rab7, nucleolin and a marker for autophagosomes. Endogenous immunostainable spermine was primarily associated with autophagosomes. When spermine synthesis was inhibited, imported spermine appeared in Rab7-positive endosomes. When ascorbate was added, heparan sulfate and spermine were transported to the nucleus where they colocalized with nucleolin. Spermine also appeared in autophagosomes. In a pulse-chase experiment, heparan sulfate and spermine were first arrested in late endosomes by actinomycin D treatment. During the chase, when arrest was abolished, heparan sulfate and spermine were both transported to the nucleus and targeted nucleolin. In amyloid precursor protein-/--fibroblasts, ascorbate failed to induce release of heparan sulfate and spermine remained in the endosomes. We propose that cell surface glypican-1 carries spermine to the endosomes and that the released heparan sulfate carries spermine across the membrane into the cytosol and then to the nucleus.

Cytochrome b561, copper, β-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S35]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein.

Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced by hypoxia.

Fetal Rat Gubernaculum Mesenchymal Cells Adopt Myogenic and Myofibroblast-Like Phenotypes

Gubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex. Embryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle. PAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling withCellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle. Our data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.

Suppression of glypican-1 autodegradation by NO-deprivation correlates with nuclear accumulation of amyloid beta in normal fibroblasts.

Heparan sulfate (HS)-containing, S-nitrosylated (SNO) glypican-1 (Gpc-1) releases anhydromannose-containing HS (anMan-HS) by SNO-catalyzed autodegradation in endosomes. Transport of anMan-HS to the nucleus requires processing of the amyloid precursor protein (APP) to amyloid beta peptides (Aβ). To further examine the relationship between APP and Gpc-1 processing in normal fibroblasts we have suppressed Gpc-1 autodegradation by aminoguanidine inhibition of NO synthesis and prevented lysosomal degradation of anMan-HS by using chloroquine. Deconvolution immunofluorescence microscopy and SDS-PAGE using anMan- and APP/Aβ-specific antibodies and markers for nuclei and autophagosomes were used to identify subcellular localization of Aβ and its oligomeric state. Wild-type mouse embryonic fibroblasts (WT MEF) grown during NO-deprivation accumulated 95-98% of Aβ as oligomers in the nucleus. WT MEF treated with chloroquine accumulated both anMan-HS and Aβ, first in the nucleus then in autophagosomes. Maximal nuclear anMan-HS and Aβ accumulation was obtained after 4 and 7 h of growth, respectively. Both yielded similar banding patterns on SDS-PAGE which were also similar to the Aβ oligomers obtained after NO-deprivation. Nuclear Aβ accumulation was marginally increased (from 54 to 58%) by suppression of both release and degradation of anMan-HS. Nuclear exit of Aβ, accumulated during growth in aminoguanidine, was enhanced by ascorbate-induced reactivation of anMan-HS production. Transgenic Alzheimer disease mouse (Tg2576) MEF, which produces excess amount of Aβ was used for comparison. Overall, nuclear Aβ exit and lysosomal degradation was compromised by inhibition of the autophagosome-lysosome pathway in both WT and Tg2576 MEF, while only WT MEF was sensitive to suppression of Gpc-1 autodegradation.

Rapid nuclear transit and impaired degradation of amyloid β and glypican-1-derived heparan sulfate in Tg2576 mouse fibroblasts.

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from S-nitrosylated glypican-1 is generated in endosomes by an endogenously or ascorbate induced S-nitrosothiol-catalyzed reaction. Expression and processing of amyloid precursor protein (APP) is required to initiate formation and endosome-to-nucleus translocation of anMan-containing HS in wild-type mouse embryonic fibroblasts (WT MEF). HS is then transported to autophagosomes and finally degraded in lysosomes. To investigate how APP-derived amyloid β (Aβ) peptide affects intracellular trafficking of HS, we have studied nuclear transit as well as autophagosome/lysosome targeting and degradation in transgenic Alzheimer disease mouse (Tg2576) MEF which produce increased amounts of Aβ. Deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody showed anMan staining in the nuclei of Tg2576 MEF after 5 min of ascorbate treatment and after 15 min in WT MEF. There was also greater nuclear accumulation of HS in Tg2576 MEF as determined by (35)S-sulfate-labeling experiments. Tg2576 MEF was less sensitive to inhibition of NO production and copper-chelation than WT MEF. By using APP- and Aβ-recognizing antibodies, we observed nuclear translocation of Aβ peptide in Tg2576 MEF but not in WT MEF. HS remained in the nucleus of WT MEF for at least 8 h and was then transported to autophagosomes. By 8 h, HS had disappeared from the nuclei of Tg2576 MEF but colocalized poorly with the autophagosome marker LC3. Aβ also disappeared rapidly from the nuclei of Tg2576 MEF. Initially, it appeared in acidic vesicles and later it accumulated extracellularly. Thus, in Tg2576 MEF there is nuclear accumulation as well as secretion of Aβ and impaired degradation of HS.

An epichromatin epitope: persistence in the cell cycle and conservation in evolution.

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.

Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiolcatalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β(A) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as (35)S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP(-/-) and APLP2(-/-) MEFs. Transfection of APP(-/-) cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate- free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.

An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a “memory” of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name “epichromatin”. We describe an “epichromatin hypothesis”, suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.

Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane

Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.

Structure of the Eps15–stonin2 complex provides a molecular explanation for EH-domain ligand specificity

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.

Alternative promoter usage and splicing of ZNF74 multifinger gene produce protein isoforms with a different repressor activity and nuclear partitioning.

We have previously shown that ZNF74, a candidate gene for DiGeorge syndrome, encodes a developmentally expressed zinc finger gene of the Kruppel-associated box (KRAB) multifinger subfamily. Using RACE, RT-PCR, and primer extension on human fetal brain and heart mRNAs, we here demonstrate the existence of six mRNA variants resulting from alternative promoter usage and splicing. These transcripts encode four protein isoforms differing at their N terminus by the composition of their KRAB motif. One isoform, ZNF74-I, which corresponds to the originally cloned cDNA, was found to be encoded by two additional mRNA variants. This isoform, which contains a KRAB motif lacking the N terminus of the KRAB A box, was devoid of transcriptional activity. In contrast, ZNF74-II, a newly identified form of the protein that is encoded by a single transcript and contains an intact KRAB domain with full A and B boxes, showed strong repressor activity. Deconvolution immunofluorescence microscopy using transfected human neuroblastoma cells and nonimmortalized HS68 fibroblasts revealed a distinct subcellular distribution for ZNF74-I and ZNF74-II. In contrast to ZNF74-I, which largely colocalizes with SC-35 in nuclear speckles enriched in splicing factors, the transcriptionally active ZNF74-II had a more diffuse nuclear distribution that is more characteristic of transcriptional regulators. Taken with the previously described RNA-binding activity of ZNF74-I and direct interaction with a hyperphosphorylated form of the RNA polymerase II participating in pre-mRNA processing, our results suggest that the two ZNF74 isoforms exert different or complementary roles in RNA maturation and in transcriptional regulation.

Traffic Pattern of Cystic Fibrosis Transmembrane Regulator through the Early Exocytic Pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.