Cellular and subcellular localization of PKMζ

In contrast to protein kinases that participate in long-term potentiation (LTP) induction and memory consolidation, the autonomously active atypical protein kinase C isoform, protein kinase Mzeta (PKMζ), functions in the core molecular mechanism of LTP maintenance and long-term memory storage. Here, using multiple complementary techniques for light and electron microscopic immunolocalization, we present the first detailed characterization of the cellular and subcellular distribution of PKMζ in rat hippocampus and neocortex. We find that PKMζ is widely expressed in forebrain with prominent immunostaining in hippocampal and neocortical grey matter, and weak label in white matter. In hippocampal and cortical pyramidal cells, PKMζ expression is predominantly somatodendritic, and electron microscopy highlights the kinase at postsynaptic densities and in clusters within spines. In addition, nuclear label and striking punctate immunopositive structures in a paranuclear and dendritic distribution are seen by confocal microscopy, occasionally at dendritic bifurcations. PKMζ immunoreactive granules are observed by electron microscopy in cell bodies and dendrites, including endoplasmic reticulum. The widespread distribution of PKMζ in nuclei, nucleoli and endoplasmic reticulum suggests potential roles of this kinase in cell-wide mechanisms involving gene expression, biogenesis of ribosomes and new protein synthesis. The localization of PKMζ within postsynaptic densities and spines suggests sites where the kinase stores information during LTP maintenance and long-term memory.     

The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.     

The cellular and subcellular localization of neuroglobin and cytoglobin -- a clue to their function?

Neuroglobin and cytoglobin are recently discovered respiratory proteins of vertebrates with yet ill-defined physiological functions. Neuroglobin is widely expressed in neurons, but not glia, in the vertebrate central and peripheral nervous systems. Other major expression sites are the retina and endocrine tissues. This distribution is indicative of a function of neuroglobin in metabolically most active, oxygen-consuming cell types, but does not yet allow to safely distinguish between different cellular roles, such as oxygen homeostasis, scavenging of reactive oxygen species or sustaining energy metabolism. Cytoglobin is predominantly expressed in connective tissue fibroblasts and related cell types in the body organs. Its main function may therefore be related to the specific amounts of extracellular matrix. Cytoglobin may hypothetically be involved in the oxygen-consuming maturation of collagen proteins. Cytoglobin is also expressed in distinct cell types of brain and retina. Its distribution strikingly differs from neuroglobin, suggesting an independent, yet unknown function.     

Adaptation or exaptation? The case of the human hand

A controversy of relevance to the study of biological form involves the concept of adaptation. This controversy is illustrated by the structure and function of the human hand. A review of the principal definitions of adaptation points to two main problems: (1) they are qualitative and make reference to the whole structure (or substructural feature) and (2) they are based on the idea of natural selection as a moulding factor. The first problem would be solved by a definition that encompasses quantitative measures of the effects of selection, drawing on new advances in the comparative method. The second problem is deeper and presents greater conceptual difficulties. I will argue that the idea of natural selection as a moulding factor depends on the notion of a genetic program for development. But regarding the hand, experimental evidence on limb development challenges the idea of a genetic program for skeletal pattern formation, undermining a simple application of standard adaptationist concepts. These considerations lead to a revised definition of adaptation and interpretation of the evolutionary determinants of the hand’s form.     

The subcellular localization of glutamate dehydrogenase (GDH): is GDH a marker for mitochondria in brain?

Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial faction, while the activities of other mitochondrial enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehydrogenase complex) were enriched only in the mitochondrial fraction. Immunoblots using polyclonal antibodies against bovine liver GDH confirmed the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubilized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubizeded only by elevated concentrations of detergents (0.3% each) plus KCl (>150mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated.     

Flavonoids: True or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase

Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC₅₀ values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.     

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells: purification, characterization and subcellular localization

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells was purified to homogeneity as judged by SDS-PAGE and silver staining of a single 32 kD band. A second, more hydrophobic product of the purification protocol contained additional bands at 45 and 80 kD. The 17β-estradiol dehydrogenase activities of both products exceeded those for 17-one reduction by more than 260-fold. Activities of 3-, 3β- and 20-dehydrogenases were absent in either fraction. Polyclonal and monoclonal antibodies raised against the 32 kD protein and the more hydrophobic product precipitated the enzymatic activity and reacted with the 32 and 80 kD bands, but not with the 45 kD band in Western blots. The subcellular localization of the enzyme was studied in sections of intact cells and of isolated organelles using gold sol coated with F(ab′)₂ fragments of monoclonal antibody F1. Gold particles were found exclusively over cytoplasmic vesicles of 120–150 nm diameter with electron-dense contents.     

Distinct subcellular localization of β-tubulin epitopes in the adult mouse brain

 A panel of monoclonal antibodies specific of α-tubulin (TU-01, TU-09) and β-tubulin (TU-06, TU-13) subunits was used to study the location of N-terminal structural domains of tubulin in adult mouse brain. The specificity of antibodies was confirmed b immunoblotting experiments. Immunohistochemical staining of vibratome sections from cerebral cortex, cerebellum, hippocampus, and corpus callosum showed that antibodies TU-01, TU-09, and TU13 reacted with neuronal and glial cells and their processes, whereas the TU-06 antibody stained only the perikarya. Dendrites and axons were either unstained or their staining was very weak. As the TU-06 epitope is located on the N-terminal structural domain of β-tubulin, the observed staining pattern cannot be interpreted as evidence of a distinct subcellular localization of β-tubulin isotypes or known post-translational modifications. The limited distribution of the epitope could, rather, reflect differences between the conformations of tubulin molecules in microtubules of somata and neurites or, alternatively, a specific masking of the corresponding region on the N-terminal domain of β-tubulin by interacting protein(s) in dendrites and axons. Accepted: 11 November 1996     

The lantibiotic nisin, a special case or not?

Nisin is a 34-residue-long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The presence of dehydrated residues and lanthionine rings (thioether bonds) in nisin, imposing structural restrains on the peptide, make it an interesting case for studying the mode of action. In addition, the relatively high activity (nM range) of nisin against Gram-positive bacteria indicates that nisin may be a special case in the large family of pore-forming peptides antibiotics. In this review, we attempted to dissect the mode of action of nisin concentrating on studies that used model membranes or biological membranes. The picture that emerges suggests that in model membrane systems, composed of only phospholipids, nisin behaves similar to the antimicrobial peptide magainin, albeit with an activity that is much lower as compared to its activity towards biological membranes. This difference can be contributed to a missing factor which nisin needs for its high activity. Novel results have identified the factor as Lipid II, a precursor in the bacterial cell wall synthesis. The special high affinity interaction of nisin with Lipid II resulting in high activity and the active role of Lipid II in the pore-formation process make nisin a special case.     

The curious case of NG2 cells: transient trend or game changer?

It has been 10 years since the seminal work of Dwight Bergles and collaborators demonstrated that NG2 (nerve/glial antigen 2)-expressing oligodendrocyte progenitor cells (NG2 cells) receive functional glutamatergic synapses from neurons (Bergles et al., 2000), contradicting the old dogma that only neurons possess the complex and specialized molecular machinery necessary to receive synapses. While this surprising discovery may have been initially shunned as a novelty item of undefined functional significance, the study of neuron-to-NG2 cell neurotransmission has since become a very active and exciting field of research. Many laboratories have now confirmed and extended the initial discovery, showing for example that NG2 cells can also receive inhibitory GABAergic synapses (Lin and Bergles, 2004) or that neuron-to-NG2 cell synaptic transmission is a rather ubiquitous phenomenon that has been observed in all brain areas explored so far, including white matter tracts (Kukley et al., 2007; Ziskin et al., 2007; Etxeberria et al., 2010). Thus, while still being in its infancy, this field of research has already brought many surprising and interesting discoveries, and has become part of a continuously growing effort in neuroscience to re-evaluate the long underestimated role of glial cells in brain function (Barres, 2008). However, this area of research is now reaching an important milestone and its long-term significance will be defined by its ability to uncover the still elusive function of NG2 cells and their synapses in the brain, rather than by its sensational but transient successes at upsetting the old order established by neuronal physiology. To participate in the effort to facilitate such a transition, here we propose a critical review of the latest findings in the field of NG2 cell physiology – discussing how they inform us on the possible function(s) of NG2 cells in the brain – and we present some personal views on new directions the field could benefit from in order to achieve lasting significance.     

Distinct subcellular localization of three isoforms of insulinoma-associated protein 2β in neuroendocrine tissues

AimsInsulinoma-associated protein 2β (IA-2β) is considered to play a significant role in regulated secretion. Recent studies have shown that the mouse brain expresses three major isoforms of IA-2β, named IA-2β60, IA-2β64, and IA-2β71. In this study, we analyzed the tissue-, cell- and organelle-specific distributions of IA-2β isoforms in mice.Main methodsTo localize IA-2β expression in mouse tissues and cells, western blot and immunohistochemical analyses were carried out. The subcellular distribution of IA-2β isoforms was assessed by sedimentation of mouse brain homogenates in a discontinuous sucrose density gradient.Key findingsIA-2β60 was abundant in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, and pituitary, and in the muscular and mucosal layers of the digestive organs. In contrast, the expression of IA-2β64 and IA-2β71 was restricted to the cerebrum, cerebellum, medulla oblongata, and pituitary, and the muscular layers of the digestive organs. Immunohistochemical analysis of mouse pancreatic islets revealed that pancreatic beta cells expressed IA-2β60 exclusively, whereas alpha and delta cells expressed all three isoforms. By the sedimentation of mouse brain homogenates, it was shown that IA-2β64 and IA-2β71 were co-localized with IA-2 on secretory granules, but were absent from synaptic vesicles (SVs). On the other hand, IA-2β60 was co-localized with synaptophisin on SVs, but was absent from secretory granules.SignificanceThe tissue-, cell- and organelle-specific distributions of IA-2β isoforms suggest that IA-2β60 has a role in secretion from SVs, whereas IA-2β64 and IA-2β71 are involved in secretion from secretory granules.     

The amino-terminus of nitric oxide sensitive guanylyl cyclase α₁ does not affect dimerization but influences subcellular localization

Background

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β₁-subunit. A splice variant (C-α₁) of the α₁-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α₁-subunit are mandatory for quantitative heterodimerization implying that the C-α₁-splice variant should lose its capacity to dimerize quantitatively.

Methodology/Principal Findings

In the current study we demonstrate preserved quantitative dimerization of the C-α₁-splice by co-purification with the β₁-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β₁-subunit and the α₁-subunit or the C-α₁ variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α₁/β₁ and C-α₁/β₁ than the negative control. In addition we show that lack of the amino-terminus in the α₁ splice variant directs it to a more oxidized subcellular compartment.

Conclusions/Significance

We conclude that the amino-terminus of the α₁-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.