Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser¹⁹-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (K_d ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S_0.5 = 25–58 μm (TH-(1–43)) and S_0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S_0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser¹⁹-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis.     

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.Disease is triggered by defects in any of 15 mouse gene products, which arepart of five distinct cytosolic molecular complexes: AP-3, homotypic fusionand vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecularassociations of these complexes, we used in vivo cross-linkingfollowed by purification of cross-linked AP-3 complexes and mass spectrometricidentification of associated proteins. AP-3 was co-isolated with BLOC-1,BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).We previously reported that this membrane-anchored enzyme is a regulator ofAP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,Salazar, G., and Faundez, V. (2008) Mol. Biol.Cell19,1415-1426). Using cells deficientin different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion intoAP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartitecomplex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1complex modulates the association of PI4KIIα with AP-3. These resultssuggest that AP-3 and BLOC-1 act, either in concert or sequentially, tospecify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are eachdefined by unique lipid and protein composition. Vesicle carriers communicateand maintain the composition of these organelles(2). Consequently defining themachineries that specify vesicle formation, composition, and delivery arecentral to understanding membrane protein traffic. Generally vesiclebiogenesis uses multiprotein cytosolic machineries to select membranecomponents for inclusion in nascent vesicles(2,3). Heterotetrameric adaptorcomplexes (AP-1 to AP-4) are critical to generate vesicles of specificcomposition from the different organelles constituting the exocytic andendocytic routes(2-4).The best understood vesicle formation machinery in mammalian cells is theone organized around the adaptor complex AP-2(5). This complex generatesvesicles from the plasma membrane using clathrin. Our present detailedunderstanding of AP-2 vesicle biogenesis mechanisms and interactions emergedfrom a combination of organellar and in vitro binding proteomicsanalyses together with the study of binary interactions in cell-free systems(5-9).In contrast, the vesicle biogenesis pathways controlled by AP-3 are far lessunderstood. AP-3 functions to produce vesicles that traffic selected membraneproteins from endosomes to lysosomes, lysosome-related organelles, or synapticvesicles(10-13).AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome(HPS;³ OnlineMendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15mouse or eight human genes trigger a common syndrome. This syndromeencompasses defects that include pigment dilution, platelet dysfunction,pulmonary fibrosis, and occasionally neurological phenotypes(14,15). All forms of HPS showdefective vesicular biogenesis or trafficking that affects lysosomes,lysosome-related organelles (for example melanosomes and platelet densegranules), and, in some of them, synaptic vesicles(11-13).Most of the 15 HPS loci encode polypeptides that assemble into five distinctmolecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,2, and 3 (14). Recently binaryinteractions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested thatarrangements of these complexes could regulate membrane protein targeting(16). Despite the abundance ofgenetic deficiencies leading to HPS and genetic evidence that HPS complexesmay act on the same pathway in defined cell types(17), we have only a partialpicture of protein interactions organizing these complexes and how they mightcontrol membrane protein targeting.In this study, we took advantage of cell-permeant and reversiblecross-linking of HPS complexes followed by their immunoaffinity purificationto identify novel molecular interactions. Cross-linked AP-3 co-purified withBLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. Wepreviously identified PI4KIIα as a cargo and regulator of AP-3recruitment to endosomes (1,18). Using mutant cellsdeficient in either individual HPS complexes or a combination of them, wefound that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Ourstudies demonstrate that subunits of four of the five HPS complexes co-isolatewith AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complexas demonstrated by sequential co-immunoprecipitations as well as by similarLAMP1 distribution phenotypes induced by down-regulation of components of thistripartite complex. Our findings indicate that BLOC-1 complex modulates therecognition of PI4KIIα by AP-3. These data suggest that AP-3, either inconcert or sequentially with BLOC-1, participates in the sorting of commonmembrane proteins along the endocytic route.     

Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation

The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1 mg/l each of benzylaminopurine (BA) and α-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.     

Partitioning of functional diversity reveals the scale and extent of trait convergence and divergence

Questions: Trait differentiation among species occurs at different spatial scales within a region. How does the partitioning of functional diversity help to identify different community assembly mechanisms? Location: Northeastern Spain. Methods: Functional diversity can be partitioned into within‐community (α) and among‐communities (β) components, in analogy to Whittaker's classical α and β species diversity concept. In light of ecological null models, we test and discuss two algorithms as a framework to measure α and β functional diversity (the Rao quadratic entropy index and the variance of trait values). Species and trait (specific leaf area) data from pastures under different climatic conditions in NE Spain are used as a case study. Results: The proposed indices show different mathematical properties but similarly account for the spatial components of functional diversity. For all vegetation types along the climatic gradient, the observed α functional diversity was lower than expected at random, an observation consistent with the hypothesis of trait convergence resulting from habitat filtering. On the other hand, our data exhibited a remarkably higher functional diversity within communities compared to among communities (α?β). In contrast to the high species turnover, there was a limited functional diversity turnover among communities, and a large part of the trait divergence occurred among coexisting species. Conclusions: Partitioning functional diversity within and among communities revealed that both trait convergence and divergence occur in the formation of assemblages from the local species pool. A considerable trait convergence exists at the regional scale in spite of changes in species composition, suggesting the existence of ecological redundancy among communities.     

Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes

The basic features of the active filaments that use nucleotide hydrolysis to organise the cytoplasm are remarkably similar in the majority of all cells and are either actin-like or tubulin-like. Nearly all prokaryotic cells contain at least one form of FtsZ, the prokaryotic homologue of tubulin and some bacterial plasmids use tubulin-like TubZ for segregation. The other main family of active filaments, assembled from actin-like proteins, occurs in a wide range of bacterial species as well as in all eukaryotes. Some bacterial plasmids also use ParM, another actin-like protein. Higher-order filament structures vary from simple to complex depending on the cellular application. Equally, filament-associated proteins vary greatly between species and it is not possible currently to trace their evolution from prokaryotes to eukaryotes. This lack of similarity except in the three-dimensional structures and longitudinal interactions between the filament subunits hints that the most basic cellular function of the filaments is to act as linear motors driven by assembly dynamics and/or bending and hence we term these filament systems 'cytomotive'. The principle of cytomotive filaments seems to have been invented independently for actin- and tubulin-like proteins. Prokaryotes appear to have a third class of cytomotive filaments, typically associated with surfaces such as membranes or DNA: Walker A cytoskeletal ATPases (WACA). A possible evolutionary relationship of WACAs with eukaryotic septins is discussed.     

Double-tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability

Bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), ubiquitous contaminants from oil and coal, is typically limited by poor accessibility of the contaminant to the bacteria. In order to measure PAH availability in complex systems, we designed a number of diffusion-based assays with a double-tagged bacterial reporter strain Burkholderia sartisoli RP037-mChe. The reporter strain is capable of mineralizing phenanthrene (PHE) and induces the expression of enhanced green fluorescent protein (eGFP) as a function of the PAH flux to the cell. At the same time, it produces a second autofluorescent protein (mCherry) in constitutive manner. Quantitative epifluorescence imaging was deployed in order to record reporter signals as a function of PAH availability. The reporter strain expressed eGFP proportionally to dosages of naphthalene or PHE in batch liquid cultures. To detect PAH diffusion from solid materials the reporter cells were embedded in 2 cm-sized agarose gel patches, and fluorescence was recorded over time for both markers as a function of distance to the PAH source. eGFP fluorescence gradients measured on known amounts of naphthalene or PHE served as calibration for quantifying PAH availability from contaminated soils. To detect reporter gene expression at even smaller diffusion distances, we mixed and immobilized cells with contaminated soils in an agarose gel. eGFP fluorescence measurements confirmed gel patch diffusion results that exposure to 2–3 mg lampblack soil gave four times higher expression than to material contaminated with 10 or 1 (mg PHE) g⁻¹.     

Altered expression of T cell Immunoglobulin-Mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis

Background

Activated T helper (Th)-1 pulmonary CD4⁺ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.

Methods

We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4⁺ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.

Results

A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren''s syndrome, BALF CD4⁺ T cells from non- Löfgren''s patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren''s patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren''s syndrome.

Conclusion

These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren''spatients is in agreement with an exaggerated Th1 response in these patients.