Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.Fluorescent proteins have become invaluable probes for studying molecular processes in living cells with light microscopy techniques (1–3). Proteins, organelles, and entire cells can be selectively visualized using a variety of fluorescent proteins fused to the proteins of interest (1–6). Combined with genetics and molecular biology techniques fluorescence microscopy provides an efficient tool for observing molecular phenotypes useful for dissecting the pathways of cell cycle progression and cell response to internal and external signals (7). However, understanding the mechanism controlling the properties of proteins in cells can be a challenging task, frequently requiring a comprehensive characterization of the proteins at the molecular level.The proteins tagged with green fluorescent protein (GFP)¹ can be also purified using GFP antibodies. Cheeseman and Desai (8) and Cristea et al. (9) have enriched GFP-tagged proteins and protein complexes for further detailed analysis by MS. The MS-based methods for protein analysis are fast, sensitive, and able to identify both proteins in complex protein mixtures and residues bearing post-translational modifications (10, 11). Thus, the addition of affinity purification and mass spectrometry steps enabled the researchers to study protein interactions and the post-translational modifications in the context of the protein subcellular localization. Juxtaposition of the protein localization, composition of the protein complexes, and post-translational modifications frequently yield a unique perspective of the cellular processes and the molecular mechanisms of their regulation (12, 13).Using fluorescent proteins also as affinity probes can be problematic in several instances. First of all, the good quality antibodies against the rapidly increasing number of fluorescent proteins (3, 6) are not yet readily available. Furthermore raising antibodies specifically recognizing fluorescent proteins originating from the same organism but fluorescing a different color can be difficult or even impossible because such proteins frequently differ by mutations of only a few amino acids (1–6). Thus, we seek an alternative approach to the design of tags suitable for subcellular localization and purification of proteins and protein complexes that is 1) independent of the availability of antibody to a specific form of a fluorescent protein, 2) suitable for multiplexing, i.e. simultaneous observation of subcellular localization of several proteins and affinity purification of the proteins and stably associated protein complexes, and 3) flexible and easy to modify to incorporate better versions of fluorescent proteins and affinity tags after they are discovered.One possible solution that satisfies the stated requirements is to use a modular tag containing a version of a fluorescent protein fused to an affinity epitope. In this case we can decouple requirements for both modules and optimize the performance of each one independently for fluorescence microscopy and affinity purification experiments. To our knowledge, this possibility was first realized by Thorn and co-worker (14) who have fused 3HA (three repeats of YPYDVPDYA epitope from hemagglutinin protein) and 13MYC (13 repeats of EQKLISEEDL epitope, corresponding to a stretch of the C-terminal amino acids of the human c-MYC protein) tags to several variants of fluorescent proteins. The authors have argued that the fusion of the fluorescent proteins to the affinity epitopes may enable fluorescence and immunochemical analysis but did not test this idea. Cheeseman and Desai (8) fused the S-peptide and hexahistidine epitopes to the GFP protein to enable additional tandem purification steps. Su and co-workers (15) also fused a hexahistidine tag (His₆) to GFP to purify recombinantly produced proteins. Although hexahistidine tag performs well for isolation of overexpressed recombinant proteins, it works poorly for affinity purification of low abundance, endogenously expressed proteins (16). A double affinity tag containing a single MYC epitope and hexahistidine was also used to purify recombinantly produced fluorescent proteins (6).Here we describe the design and implementation of the modular fluorescent and affinity tags. These tags contain a variety of fluorescent proteins, which can be used exclusively for obtaining subcellular visualization, and several small epitope tags that can be utilized to perform two-step affinity purification. To test the performance of the constructs produced, we tagged two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, with a variety of modular tags.Eisosomes are large heterodimeric protein complexes recently discovered in Saccharomyces cerevisiae (17). There are ∼50–100 eisosomes in each mature yeast cell distributed uniformly in a characteristic dotted pattern at the cell surface periphery. Each eisosome contains ∼2000–5000 copies of Pil1 and Lsp1. It was shown that eisosomes serve as portals of endocytosis in yeast. The function of eisosomes is regulated by reversible phosphorylation (18, 19).Among the constructs tested, we found several combinations of fluorescent protein and affinity tags that were optimal for determining subcellular localization and purification of the proteins and protein complexes. We applied these tags to further investigate eisosomes and found several new protein components of the complexes and obtained new insights into molecular mechanisms regulating eisosome integrity by reversible phosphorylation and dephosphorylation. Our results indicate that an approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.     

Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.     

High‐Temperature Shock Enabled Nanomanufacturing for Energy‐Related Applications

Functional nanomaterials are playing a crucial role in the emerging field of energy‐related devices. Recently, as a novel synthesis method, high‐temperature shock (HTS), which is rapid, low cost, eco‐friendly, universal, scalable, and controllable, has provided a promising option for the rational design and synthesis of various high‐quality nanomaterials. In this report, the HTS technique, including the equipment setup and operating principle, is systematically introduced, and recent progress in the synthesis of nanomaterials for energy storage and conversion applications using this HTS method is summarized. The growth mechanisms of nanoparticles and carbonaceous nanomaterials are thoroughly discussed, followed by the summary of the characteristic advantages of the HTS strategy. A series of nanomaterials prepared by the HTS method, including carbon‐based films, metal nanoparticles and compound nanoparticles, show high performance in the diverse applications of storage energy batteries, highly active catalysts, and smart energy devices. Finally, the future perspectives and directions of HTS in nanomanufacturing for broader applications are presented.     

The Immunoregulation Effect of Alpha 1-Antitrypsin Prolong β-Cell Survival after Transplantation

Islet transplantation has considerable potential as a cure for diabetes. However, the difficulties that arise from inflammation and the immunological rejection of transplants must be addressed for islet transplantation to be successful. Alpha 1-antitrypsin (AAT) inhibits the damage on β cells caused by inflammatory reactions and promotes β-cell survival and proliferation. This protein also induces specific immune tolerance to transplanted β cells. However, whether the expression of AAT in β cells themselves could eliminate or decrease immunological rejection of transplants is not clear. Therefore, we established a β cell line (NIT-hAAT) that stably expresses human AAT. Interestingly, in a cytotoxic T lymphocyte (CTL)-killing assay, we found that hAAT reduced apoptosis and inflammatory cytokine production in NIT-1 cells and regulated the Th1/Th2 cytokine balance in vitro. In vivo transplantation of NIT-hAAT cells into mice with diabetes showed hAAT inhibited immunological rejection for a short period of time and increased the survival of transplanted β cells. This study demonstrated that hAAT generated remarkable immunoprotective and immunoregulation effects in a model of β cell islet transplantation for diabetes model.     

Association of Plasma IL-6 and Hsp70 with HRV at Different Levels of PAHs Metabolites

Background

Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with reduced heart rate variability (HRV), a strong predictor of cardiovascular diseases, but the mechanism is not well understood.

Objectives

We hypothesized that PAHs might induce systemic inflammation and stress response, contributing to altered cardiac autonomic function.

Methods

HRV indices were measured using a 3-channel digital Holter monitor in 800 coke oven workers. Plasma levels of interleukin-6 (IL-6) and heat shock protein 70 (Hsp70) were determined using ELISA. Twelve urinary PAHs metabolites (OH-PAHs) were measured by gas chromatography-mass spectrometry.

Results

We found that significant dose-dependent relationships between four urinary OH-PAHs and IL-6 (all P _trend<0.05); and an increase in quartiles of IL-6 was significantly associated with a decrease in total power (TP) and low frequency (LF) (P _trend = 0.014 and 0.006, respectively). In particular, elevated IL-6 was associated in a dose-dependent manner with decreased TP and LF in the high-PAHs metabolites groups (all P _trend<0.05), but not in the low-PAHs metabolites groups. No significant association between Hsp70 and HRV in total population was found after multivariate adjustment. However, increased Hsp70 was significantly associated with elevated standard deviation of NN intervals (SDNN), TP and LF in the low-PAHs metabolites groups (all P _trend<0.05). We also observed that both IL-6 and Hsp70 significantly interacted with multiple PAHs metabolites in relation to HRV.

Conclusions

In coke oven workers, increased IL-6 was associated with a dose-response decreased HRV in the high-PAHs metabolites groups, whereas increase of Hsp70 can result in significant dose-related increase in HRV in the low-PAHs metabolites groups.     

Phylogeny of Prokaryotes and Chloroplasts Revealed by a Simple Composition Approach on All Protein Sequences from Complete Genomes Without Sequence Alignment

The complete genomes of living organisms have provided much information on their phylogenetic relationships. Similarly, the complete genomes of chloroplasts have helped to resolve the evolution of this organelle in photosynthetic eukaryotes. In this paper we propose an alternative method of phylogenetic analysis using compositional statistics for all protein sequences from complete genomes. This new method is conceptually simpler than and computationally as fast as the one proposed by Qi et al. (2004b) and Chu et al. (2004). The same data sets used in Qi et al. (2004b) and Chu et al. (2004) are analyzed using the new method. Our distance-based phylogenic tree of the 109 prokaryotes and eukaryotes agrees with the biologists tree of life based on 16S rRNA comparison in a predominant majority of basic branching and most lower taxa. Our phylogenetic analysis also shows that the chloroplast genomes are separated to two major clades corresponding to chlorophytes s.l. and rhodophytes s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution.Reviewing Editor: Dr. John Oakeshott     

Synthesis and biological evaluation of 4,4-dimethyl lithocholic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B

Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of both insulin and leptin signals. For years, inhibiting of PTP1B has been considered to be a potential therapeutics for treating Type 2 diabetes and obesity. Recently, we recognized lithocholic acid (LCA) as a natural inhibitor against PTP1B (IC₅₀ = 12.74 μM) by a vertical screen for the first time. Further SAR research was carried out by synthesizing and evaluating a series of compounds bearing two methyls at C-4 position and a fused heterocycle to ring A. Among them, compound 14b achieved a PTP1B inhibitory activity about eightfold than LCA and a 14-fold selectivity over the homogenous enzyme TCPTP.     

Structural analysis of chloroplast tail‐anchored membrane protein recognition by ArsA1

In mammals and yeast, tail‐anchored (TA) membrane proteins destined for the post‐translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well‐known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane‐trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide‐free open state and bound to adenylyl‐imidodiphosphate. This approximately 80‐kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA‐binding groove comprise the interlocking hook‐like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.