Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy

The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy.     

Alternative Translation Initiation Generates Cytoplasmic Sheep Prion Protein

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein (^GFPPrP) in N2a cells, with variable sequence context surrounding the start codon Met¹. ^GFPPrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of ^GFPPrP were detected intracellularly, starting in frame from Met¹⁷. When ^GFPPrP was expressed with a compromised Kozak sequence (^GFPPrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of ^GFPPrP*, whereas the N-terminal fragments starting from Met¹⁷ were still present. Formation of these N-terminal fragments was completely abolished when Met¹⁷ was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met¹⁷ is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.PrP^{C 2} is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases (1, 2). According to the prion hypothesis, a misfolded, pathogenic form of the protein (PrP^Sc) is the sole constituent of transmissible prions (3, 4), but the molecular details and required environs for the misfolding are incompletely understood. As would be expected for a glycosylphosphatidylinositol-anchored protein with N-linked glycans, PrP^C is observed at the outer leaflet of the plasma membrane, the end point of the secretory route. The half-time at the plasma membrane is fairly short, because the protein may undergo shedding or endocytic internalization (5–9). Thus, PrP^C can be encountered throughout the secretory and endocytic routes and is also able to leave cells via exosomes derived from multivesicular endosomes (10). In agreement with this, studies of the subcellular distribution of PrP^C in mammalian brain have identified localization to the outer cell membrane, in the Golgi apparatus, and in endosomal vesicles (11, 12). However, others have found that PrP^C is not solely associated with membranes, but, in some subpopulations of neurons, is localized to the cytoplasm (13, 14). In line with the latter observations, transgenic mice expressing PrP carrying a C-terminal GFP tag demonstrated intense cytoplasmic fluorescence from a limited number (approximately 1%) of the neurons in certain brain areas, such as the hippocampus (15). Immunohistochemical detection of intracellular, possibly cytoplasmic, PrP has also been reported from large mononuclear cells in the gut wall of sheep (16) and from enteric neurons in mice (17). The recent observations of pronounced cytoplasmic aggregation of PrP in pancreatic β-cells of rats prone to development of diabetes mellitus provide a perplexing example of nonstandard PrP localization in non-neuronal cells (18).The flexibility observed in the subcellular localization of PrP^C has been suggested to be a requirement for normal functions of the protein (14, 19, 20), but how cytoplasmic and nuclear variants arise has not been established. Cytoplasmic PrP could be a result of retro-translocation from the endoplasmic reticulum (ER), as part of an unfolded protein response (21–23) or from attenuated ER import of PrP under conditions of lumenal stress in the ER (24, 25). The finding of intact ER-targeting signal sequences on cytoplasmic PrPs (25, 26) favors the latter mechanism, namely a reduced ER import of PrP, possibly caused by saturation of the ER translocation machinery or an overload of unfolded proteins within the ER. However, no signs of stress or pathology could be detected in neurons of wild-type mice expressing cytoplasmic PrP (14), which led to the suggestion that the cytoplasmic appearance of PrP could constitute a physiologically relevant, but minor, pathway for the protein.Forced cytoplasmic expression of PrP in transgenic mice (22) and in the nematode Caenorhabditis elegans (27) resulted in neurodegenerative disease, suggesting that toxic mislocalization of PrP could be part of the pathogenic mechanism in prion diseases (28). However, transgenic mice expressing cytoplasmic PrP, on a PrP-null background, developed cerebellar atrophy but were resistant to experimental prion infection (29), suggesting that cytoplasmic PrP is unlikely to serve as substrate for prion replication. Furthermore, data obtained from transgenic mice expressing an anchorless secretory PrP show that, although these mice accumulate PrP-containing amyloid plaques upon challenge with PrP^Sc, they fail to develop clinical prion disease (30). Thus, membrane-attached PrP appears to be a prerequisite for development of prion-derived neurodegeneration.In eukaryotes, ribosomes bind specifically to linear mRNAs carrying a 7-methylguanosine 5′-end cap and slide along the mRNA in the 5′ → 3′ direction until they encounter the first start codon (AUG), from which the protein translation starts exclusively. Therefore, eukaryotic mRNAs are generally monocistronic. However, deviations from this standard principle have been reported, in which protein translation is initiated at alternative start codons either up or downstream from the primary AUG. The best characterized mechanism is known as context-dependent leaky ribosomal scanning (LRS) (31). This cap-dependent mechanism is particularly operative when the optimal (5′-GCCRCCaugG-3′) sequence context surrounding the first AUG codon is compromised, most notably at positions R⁻³ (where R= purine, A or G, but optimally G) and G⁺⁴ (32, 33).In this work, we report that in a cell culture system, sheep PrP mRNA displays a tendency to allow alternative translation initiation through LRS. Met¹⁷ serves as an internal in-frame alternative start codon giving rise to PrP with a severely shortened ER-targeting peptide.Although the LRS mechanism is active in sheep PrP, it appears to occur much less in mouse PrP (34). The molecular explanation and possible pathophysiological relevance of these observations in relation to PrP function await further studies. Interestingly, during the review process of this paper, observations of cytoplasmic PrP similar to some of those described herein were reported for human and hamster PrP (35).     

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.     

Comparisons of genetic parameters and clonal value predictions from clonal trials and seedling base population trials of radiata pine

Different methods for predicting clonal values were explored for diameter growth (diameter at breast height (DBH)) in a radiata pine clonal forestry program: (1) clones were analyzed with a full model in which the total genetic variation was partitioned into additive, dominance, and epistasis (Clone Only—Full Model); (2) clones were analyzed together with seedling base population data (Clone Plus Seedling (CPS)), and (3) clones were analyzed with a reduced model in which the only genetic term was the total genetic variance (Clone Only—Reduced Model). DBH was assessed at age 5 for clones and between ages 4 to 13 at the seedling trials. Significant additive, dominance, and epistatic genetic effects were estimated for DBH using the CPS model. Nonadditive genetic effects for DBH were 87% as large as additive genetic effects. Narrow-sense () and broad-sense () heritability estimates for DBH using the CPS model were 0.14 ± 0.01 and 0.26 ± 0.01, respectively. Accuracy of predicted clonal values increased 4% by combining the clone and seedling data over using clonal data alone, resulting in greater confidence in the predicted genetic performance of clones. Our results indicate that exploiting nonadditive genetic effects in clonal varieties will generate greater gains than that typically obtainable from conventional family-based forestry of radiata pine. The predicted genetic gain for DBH from deployment of the top 5% of clones was 24.0%—an improvement of more than 100% over family forestry at the same selection intensity. We conclude that it is best practice to predict clonal values by incorporating seedling base population data in the clonal analysis.     

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission

The modification of proteins by the small ubiquitin‐like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin‐related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial‐anchored protein ligase (MAPL) as the first mitochondrial‐anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.     

Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance

Background  

Occludin is a tetraspanin protein normally localized to tight junctions. The protein interacts with a variety of pathogens including viruses and bacteria, an interaction that sometimes leads to its extrajunctional localization.     

Antibody array analysis with label-based detection and resolution of protein size

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670-10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.