Crosstalk in transition: the translocation of Akt.

Akt/PKB is an important crosstalk node at the junction between a number of major signalling pathways in the mammalian cell. As a significant nutrient sensor, Akt plays a central role in many cellular processes, including cell growth, cell survival and glucose metabolism. The dysregulation of Akt signalling is implicated in the development of many diseases, from diabetes to cancer. The translocation of Akt from cytosol to plasma membrane is a crucial step in Akt activation. Akt is initially synthesized on the endoplasmic reticulum, but translocates to the plasma membrane (PM) in response to insulin stimulation, where it may be activated. The Akt is then recycled to the cytoplasm. The activated Akt may propagate signals to downstream substrates both at the PM and in the cytosol, hence understanding the translocation dynamics is an important step in dissecting the signalling system. At the present time, however, knowledge concerning the translocation of either activated and unactivated Akt is scant. Here we present a simple, deterministic, three-compartment ordinary differential equation model of Akt translocation in vitro. This model can reproduce the salient features of Akt translocation in a manner consistent with the experimental data. Furthermore, we demonstrate that this system is equivalent to a damped harmonic oscillator, and analyse the steady state and transient behaviour of the model over the entire parameter space.

Subcellular distribution of choline acetyltransferase by immunogold electron microscopy in non-neuronal cells: placenta, airways and murine embryonic stem cells.

AIMS: Acetylcholine is synthesized in more or less all mammalian cells. However, little is known about the subcellular location of acetylcholine synthesis. Therefore, in the present experiments the subcellular location of the synthesizing enzyme choline acetyltransferase (ChAT) was investigated by anti-ChAT immunogold electron microscopy in human placenta and airways as well as in a murine embryonic stem cell line (CGR8 cell line). MAIN METHODS: Human tissue was obtained as so-called surplus tissue (after delivery/surgical removal because of lung tumor); the CGR8 stem cell line was cultured under standard conditions. For human tissue a monoclonal mouse anti-ChAT antibody (ab) was used and for the CGR8 cell line a polyclonal goat anti-ChAT ab. Immunogold electron microscopy was applied to identify the subcellular location of ChAT. KEY FINDINGS: In trophoblast cells (placenta) specific anti-ChAT immunogold deposition was found within the cell membrane, microvilli, and caveolae but also within the cytosol, for example associated with intermediate filaments. In addition, immunogold deposition was identified within mitochondria and the nuclear membrane. In airway epithelial cells anti-ChAT immunogold was found particularly within the apical cell membrane, cilia, submucosa, cytosol and nuclear membrane. Likewise alveolar macrophages showed positive anti-ChAT immunogold within the nucleus, nuclear membrane and granula. Also in the CGR8 cell line positive anti-ChAT immunogold was identified within the cell nucleus and cytosol. SIGNIFICANCE: The present experiments demonstrate a wide subcellular distribution of ChAT with particular preference of the cell membrane in human epithelial cells.

Transglutaminase 2: a multi-functional protein in multiple subcellular compartments.

Transglutaminase 2 (TG2) is a multifunctional protein that can function as a transglutaminase, G protein, kinase, protein disulfide isomerase, and as an adaptor protein. These multiple biochemical activities of TG2 account for, at least in part, its involvement in a wide variety of cellular processes encompassing differentiation, cell death, inflammation, cell migration, and wound healing. The individual biochemical activities of TG2 are regulated by several cellular factors, including calcium, nucleotides, and redox potential, which vary depending on its subcellular location. Thus, the microenvironments of the subcellular compartments to which TG2 localizes, such as the cytosol, plasma membrane, nucleus, mitochondria, or extracellular space, are important determinants to switch on or off various TG2 biochemical activities. Furthermore, TG2 interacts with a distinct subset of proteins and/or substrates depending on its subcellular location. In this review, the biological functions and molecular interactions of TG2 will be discussed in the context of the unique environments of the subcellular compartments to which TG2 localizes.

Tissue distribution of mevalonate pyrophosphate decarboxylase in guinea pig.

We previously reported that mevalonate pyrophosphate decarboxylase (MPD) is located in the cytosol and that MPD level in the liver is higher than in other rat tissues. In the present study, we further investigated the tissue distribution of MPD in guinea pigs by immunoblotting using anti-rat MPD antiserum. When immunoblot analysis was carried out using guinea pig brain, the antiserum reacted with 46-kDa protein as well as a substance with the same molecular weight of MPD in mice. Protein of 46-kDa detected in guinea pig liver treated with 0.1% pravastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor indicating a liver-specific effect, was increased 3-fold as compared with nontreated guinea pigs; however, 46-kDa protein in the brain treated with pravastatin was similar to that treated without pravastatin. When the subcellular distribution of MPD in the brain, liver, kidney, and testis, was examined by cell fractionation, MPD was mostly detected in the cytosol fraction of all tissues. From these data, the 46-kDa protein was identified as MPD. Next, when the tissue distribution of MPD was examined, MPD in the liver was higher than in other tissues. The relative amount of MPD in guinea pig kidney was higher than in rats and similar to in mice, as MPD in the liver of the same species was taken as 1. Furthermore, the correlation coefficient between guinea pigs and rats or mice in the tissue distribution of MPD was 0.69 or 0.72, respectively. These data indicate a relationship in tissue distribution between guinea pigs and rats or mice, although the tissue-specific regulator of MPD between species somewhat differed.

Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion.

The glycolytic enzyme hexokinase (HK) is suggested to play a role in ischemic preconditioning (IPC). In the present study we determined how ischemic preconditioning affects HK activity and HKI and HKII protein content at five different time points and three different subcellular fractions throughout cardiac ischemia-reperfusion. Isolated Langendorff-perfused rat hearts (10 groups of 7 hearts each) were subjected to 35 min ischemia and 30 min reperfusion (control groups); the IPC groups were pretreated with 3 times 5-min ischemia. IPC was without effect on microsomal HK activity, and only decreased cytosolic HK activity at 35 min ischemia, which was mimicked by decreased cytosolic HKII, but not HKI, protein content. In contrast, mitochondrial HK activity at baseline and during reperfusion was elevated by IPC, without changes during ischemia. No effect of IPC on mitochondrial HK I protein content was observed. However, mitochondrial HK II protein content during reperfusion was augmented by IPC, albeit not following the IPC stimulus. It is concluded that IPC results in decreased cytosolic HK activity during ischemia that could be explained by decreased HKII protein content. IPC increased mitochondrial HK activity before ischemia and during reperfusion that was only mimicked by increased HK II protein content during reperfusion. IPC was without effect on the phosphorylation status of HK before ischemia. We conclude that IPC is associated with 1) a biphasic response of increased mitochondrial HK activity before and after ischemia, 2) decreased cytosolic HK activity during ischemia, and 3) cellular redistribution of HKII but not HKI.

Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments.

Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting.

Molecular cloning and characterization of a novel V-ATPase associated protein, DVA9.2, from human dendritic cells.

The vacuolar proton-ATPase (V-ATPase) is a ubiquitous ATP-driven H(+) transporter that functions in numerous cell processes. Accumulating evidence shows important roles of V-ATPase in tumor metastasis and antigen presentation of dendritic cells (DC). A novel V-ATPase associated protein, designated as DVA9.2 (dendritic cell-derived V-ATPase associated protein of 9.2 kDa), has been identified from a human DC cDNA library by large-scale random sequencing. Full length cDNA of DVA9.2 encodes an 81-residue protein that shares 70-80% homology with human V-ATPase subunit M9.2. Distant relationship is also found with Vma21p, a yeast protein required for V-ATPase assembly. DVA9.2 contains a conserved domain, ATP synthase subunit H (pafm05493), and two membrane-spanning helices. DVA9.2 mRNA is detectable in several human tumor cell lines as well as some human normal cells and tissues. Moreover, the inducible expression of DVA9.2 mRNA in DC during maturation is observed. DVA9.2 displays integration with membrane and main localization in lysosome, endoplasmic reticulum and Golgi-associated organelles, only less at the plasma membrane. In addition, DVA9.2 is co-localized with V(0)-sector subunit a. Silencing of DVA9.2 by small interfering RNA (siRNA) does not affect the V-ATPase activity in cell membrane fractions or attenuate the migration and invasion in breast cancer MDA-MB-231 cells. These results indicate that DVA9.2, as a novel V-ATPase-associated protein, is not essential for the activity of V-ATPase complex and may be involved in functions of DC.

Effects of different environmental oxygen levels on free radical processes in fish.

Changes in oxygen levels occur frequently in aquatic environments; therefore, water organisms, including fishes, evolve a wide spectrum of adaptations to both anoxia/hypoxia and hyperoxia. The review describes oxidative damage to cellular constituents by reactive oxygen species, alterations in glutathione status, and response of antioxidant enzymes to variable oxygen availability in fish. Anoxia- and hypoxia-tolerant species demonstrate an anticipatory increase of some antioxidant enzymes during low-oxygen state in order to enhance their antioxidant potential for dealing with possible oxidative stress upon return to normoxia. Under hyperoxic conditions, it seems that the glutathione system plays an important adaptive role. Most stressful conditions lead to a quick increase in lipid peroxidation products that, in turn, are detoxified rapidly by respective low- and high-molecular weight antioxidants. A scheme on possible ways of regulating antioxidant enzymes by different oxygen levels is proposed.

Effect of aging on intracellular distribution of abasic (AP) endonuclease 1 in the mouse liver.

The abasic (AP) endonuclease (APE1) plays a central role in the base excision repair (BER) pathway for repairing oxidatively damaged bases and abasic sites in mammalian genomes. We have investigated age-dependent changes in APE activity, contributed primarily by APE1, in total extracts as well as in nuclear, mitochondrial, and cytoplasmic compartments of mouse hepatocytes. The APE1 protein and mRNA levels did not differ significantly between the livers of 4-mo (young), 10-mo (middle-aged), and 20-mo (old) mice, and corresponds with similar APE activity. However, we observed a 2-fold increase in specific activity of APE1 in the nucleus, a 2-fold decrease in the cytoplasm, and a 6-fold increase in the mitochondrial matrix of hepatocytes of the old relative to the young animals. Surprisingly, in the middle-age animals we observed 30% increase in APE activity in the nucleus but 6-fold in the mitochondrial matrix. These results indicate age-dependent accumulation of APE1 in the nucleus and mitochondria. Such redistribution occurred early in the mitochondria during the aging process and preferential accumulation of APE in the nucleus was more gradual which may reflect distinct levels of oxidative stress in these organelles.

Teaching cell biology to nonscience majors through forensics, or how to design a killer course.

Nonscience majors often do not respond to traditional lecture-only biology courses. However, these students still need exposure to basic biological concepts. To accomplish this goal, forensic science was paired with compatible cell biology subjects. Several topics such as human development and molecular biology were found to fulfill this purpose. Another goal was to maximize the hands-on experience of the nonscience major students. This objective was fulfilled by specific activities such as fingerprinting and DNA typing. One particularly effective teaching tool was a mock murder mystery complete with a Grand Jury trial. Another objective was to improve students' attitudes toward science. This was successful in that students felt more confident in their own scientific abilities after taking the course. In pre/post tests, students answered four questions about their ability to conduct science. All four statements showed a positive shift after the course (p values ranging from.001 to.036, df = 23; n = 24). The emphasis on experiential pedagogy was also shown to increase critical thinking skills. In pre/post testing, students in this course significantly increased their performance on critical thinking assessment tests from 33.3% correct to 45.3% (p =.008, df = 4; n = 24).

Rice phospholipase D isoforms show differential cellular location and gene induction.

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, stress responses, protein trafficking, and membrane metabolism. This report describes the cloning and characterization of three novel PLD genes from rice, designated RPLD3, RPLD4 and RPLD5. The rice PLDs, including the previously isolated RPLD1 and RPLD2, are similar to PLD subfamilies of Arabidopsis: Based on sequence homology and domain conservation, RPLD1 is most similar to the PLDalpha subfamily of PLDs while RPLD5 most closely resembles the PLDdelta type. RPLD2, 3 and 4 represent a unique subfamily, although they are most similar to PLDalpha. RPLD1 is located on chromosome 1, RPLD5 on chromosome 3, and RPLD2, RPLD3, and RPLD4 are tandemly arrayed on chromosome 5. Transcriptional analysis reveals that RPLD1, present in healthy rice vegetative tissues, is induced rapidly but transiently in wounded leaf tissues. RPLD2, also induced by wounding, is present at lower levels but for a more prolonged duration than RPLD1. Immunolocalization with peptide specific antibodies to each of the five PLDs was used to demonstrate that the isoforms have overlapping but distinct patterns of distribution in healthy rice cells. RPLD1 was detected in mesophyll cell wall, membranes, and chloroplasts, whereas RPLD3 and RPLD4 were located predominantly in the chloroplasts. Labeling of RPLD2 and RPLD5 was sparse, and was most concentrated in the secondary walls of xylem (RPLD2) and guard cells (RPLD2 and RPLD5). This combined information on structural features, expression profiles, and cellular localization will assist the basis for dissection of PLD isoform function in rice.