Selection, Isolation, and Characterization of Cadmium-Resistant Datura innoxia Suspension Cultures

Datura innoxia cells from suspension cultures were selected for their ability to grow and divide rapidly in normally lethal concentrations of cadmium. Cells resistant to 12.5, 25, 50, 100, 160, 200, and 250 micromolar cadmium chloride were isolated and utilized to initiate cell suspension cultures resistant to this toxic metal ion. Variant cell lines retained their ability to grow in cadmium after being grown in its absence for more than 400 generations. Resistance to cadmium was correlated with the synthesis of low molecular weight, cysteine-rich, cadium-binding proteins. Synthesis of these proteins was induced rapidly in cadmium-resistant cells in response to a challenge of cadmium. Induction was detectable within one hour after exposure of the cells to the metal ion. Accumulation of protein bound cadmium reached a maximum eight to twelve hours following exposure. Metal-binding proteins were not detectable in the cadmium sensitive D. innoxia cells from which resistant cells were derived.     

Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labeling probes

The membrane proteome plays a critical role in electron transport processes in Shewanella oneidensis MR-1, a bacterial organism that has great potential for bioremediation. Biotinylation of intact cells with subsequent affinity-enrichment has become a useful tool for characterization of the membrane proteome. As opposed to these commonly used, water-soluble commercial reagents, we here introduce a family of hydrophobic, cell-permeable affinity probes for extensive labeling and detection of membrane proteins. When applied to S. oneidensis cells, all three new chemical probes allowed identification of a substantial proportion of membrane proteins from total cell lysate without the use of specific membrane isolation method. From a total of 410 unique proteins identified, approximately 42% are cell envelope proteins that include outer membrane, periplasmic, and inner membrane proteins. This report demonstrates the first application of this intact cell biotinylation method to S. oneidensis and presents the results of many identified proteins that are involved in metal reduction processes. As a general labeling method, all chemical probes we introduced in this study can be extended to other organisms or cell types and will help expedite the characterization of membrane proteomes.     

Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization

Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.     

Noble metals strip peptides from class II MHC proteins

Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a 'peptide-empty' conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.     

Asymmetric localisation of planar polarity proteins: Mechanisms and consequences

Planar polarisation of tissues is essential for many aspects of developmental patterning. It is regulated by a conserved group of core planar polarity proteins, which localise asymmetrically within cells prior to morphological signs of polarisation. A subset of these core proteins also interact across cell boundaries, mediating intercellular communication that co-ordinates polarity between neighbouring cells. Core protein localisation subsequently mediates changes in the actin cytoskeleton which lead to overt polarisation. In this review we discuss the mechanisms by which the core planar polarity proteins become asymmetrically localised, and the significance of this subcellular localisation for both intercellular communication and downstream effects on the cytoskeleton.     

Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line

A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.     

Secondary Transporters for Nickel and Cobalt Ions: Theme and Variations

Nickel/cobalt transporters (NiCoTs), a family of secondary metal transporters in prokaryotes and fungi, are characterized by an eight-transmembrane-domain (TMD) architecture and mediate high-affinity uptake of cobalt and/or nickel ions into the cells. One of the strongly conserved regions within the NiCoTs is the signature sequence RHA(V/F)DADHI within TMD II. This stretch of amino acid residues plays an important role in the affinity, velocity and specificity of metal transport. Some relatives of the NiCoTs, named HupE, UreJ and UreH, contain a similar signature sequence and are encoded within or adjacent to [NiFe] hydrogenase or urease operons, or elsewhere in the genome of many prokaryotes. HupE and UreH from Rhodopseudomonas palustris CGA009 and UreJ from Cupriavidus necator H16 were shown to mediate Ni²⁺ transport upon heterologous production in E. coli. Other variants of NiCoTs are found in many marine cyanobacteria and in plants. The cyanobacterial proteins are encoded by a segment adjacent to the genes for [Ni] superoxide dismutase and a corresponding putative maturation peptidase. The plant proteins contain N-terminal sequences resembling bipartite transit peptides of thylakoid lumenal and thylakoid integral membrane precursor proteins; expression of a YFP-fusion protein in transfected leaf cells is consistent with targeting of this protein to the plastid, but the function of the plant gene product has yet to be demonstrated.     

Selective photolabeling of proteins using photoactivatable GFP

Today's cell biologists rely on an assortment of advances in microscopy methods to study the inner workings of cells and tissues. Among these advances are fluorescent proteins which can be used to tag specifically and, in many cases, non-invasively proteins of interest within a living cell. Introduction of DNA encoding the fluorescently tagged protein of interest into a cell readily allows the visualization of the protein's localization and time-lapse imaging allows the movement of the structure or organelle to which the protein is localized to be observed. To monitor the movement of the protein within the population, researchers generally have to highlight a pool of molecules by perturbing the steady-state fluorescence. This perturbation has traditionally been performed by photobleaching the molecules within a selected region of the cell and monitoring the recovery of molecules into this region or the loss of molecules within other regions. Fluorescent proteins are now available, which allow a pool of molecules to be highlighted directly by photoactivation. Here, we discuss the technical aspects for using one of these recently developed photoactivatable fluorescent proteins, PA-GFP.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

The role of lysosomes in iron metabolism and recycling

Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, 'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.     

Use of Green Fluorescent Protein in mouse embryos

Green Fluorescent Protein (GFP) has rapidly been established as a versatile and powerful cell marker in many organisms. Initial problems in using it in mammalian cells were solved by introducing mutations to increase its solubility at higher temperatures, such that GFP has now been used as a reporter in both gene expression and cell lineage studies, and to localize proteins within mammalian cells. GFP has two unique advantages: (i) the protein becomes fluorescent in an autocatalytic reaction, so that it can be introduced into any cell type simply as a cDNA or mRNA, or as protein; (ii) it is "bright" enough to be visualized in living cells under conditions that do not cause photodamage to the cells. In this article we outline the ways in which we have used GFP mRNA and cDNA in our studies of mouse cell lineages, and to characterize the behavior of proteins within the embryos.     

Bacterial effector interplay: a new way to view effector function

Bacterial pathogens are dependent on virulence factors to efficiently colonize and propagate within their hosts. Many Gram-negative bacterial pathogens rely on specialized proteinaceous secretion systems that inject virulence factors, termed effectors, directly into host cells. These bacterial effector proteins perform various functions within host cells; however, regulation of their function within the host cell is highly enigmatic. It is becoming increasingly apparent that many of these effectors directly influence and regulate each other and their mechanisms within the host cell. We discuss the emerging theme of bacterial effector interplay impacting infection and the importance of investigating this topic.     

Mercuric chloride activates the Src-family protein tyrosine kinase, Hck in myelomonocytic cells.

Hck is a member of the Src-family of protein tyrosine kinases that appears to function in mature leukocytes to communicate a number of extracellular signals including various cytokines. In this study we show that the thiol-reactive heavy metal, mercuric chloride (HgCl2) induces rapid and robust activation of tyrosine phosphorylation within human myelomonocytic cells. This increase in tyrosine-phosphorylated proteins requires the activity of Hck because both kinase inactive alleles of Hck and pharmacological inhibitors selective for the Src-family kinases are able to abrogate the cellular response to HgCl2. Furthermore, ectopic expression of Hck in murine fibroblasts is able to confer HgCl2 responsiveness, as indicated by an increase in tyrosine-phosphorylated proteins to a normally nonresponsive cell line. Concomitant with the activation of Hck, there is a physical association of Hck with another cytoplasmic protein tyrosine kinase, Syk. The ability of HgCl2 to activate Src-family kinases such as Hck in hematopoietic cells may help explain why exposure to the heavy metal is associated with immune system dysfunction in rodents as well as humans.     

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana

In flowering plants, cell–cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane‐bound proteins associated with the endomembrane system and the cell surface. While many synergid‐expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane‐bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co‐localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI‐anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane‐associated trafficking within a haploid cell actively involved in polar transport.     

Photo-oxidation of cells generates long-lived intracellular protein peroxides

Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of rose bengal, is enhanced in D(2)O, and is decreased by azide, consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to about 1.5 nmoles peroxide per 10(6) cells, or 10 nmoles/mg cell protein, and account for up to approximately 15% of the O(2) consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular protein peroxide levels decrease with t(1/2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads to novel long-lived, but reactive, intracellular protein peroxides via singlet oxygen-mediated reactions.     

The ins and outs of host‐microsporidia interactions during invasion,proliferation and exit

Microsporidia are a large group of fungal‐related obligate intracellular parasites. They are responsible for infections in humans as well as in agriculturally and environmentally important animals. Although microsporidia are abundant in nature, many of the molecular mechanisms employed during infection have remained enigmatic. In this review, we highlight recent work showing how microsporidia invade, proliferate and exit from host cells. During invasion, microsporidia use spore wall and polar tube proteins to interact with host receptors and adhere to the host cell surface. In turn, the host has multiple defence mechanisms to prevent and eliminate these infections. Microsporidia encode numerous transporters and steal host nutrients to facilitate proliferation within host cells. They also encode many secreted proteins which may modulate host metabolism and inhibit host cell defence mechanisms. Spores exit the host in a non‐lytic manner that is dependent on host actin and endocytic recycling proteins. Together, this work provides a fuller picture of the mechanisms that these fascinating organisms use to infect their hosts.     

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer–induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10–15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.     

Studying cytoskeletal dynamics in living cells using green fluorescent protein

Microfilaments, intermediate filaments, and microtubules are three major cytoskeletal systems providing cells with stability to maintain proper shape. Although the word “cytoskeleton” implicates rigidity, it is quite dynamic exhibiting constant changes within cells. In addition to providing cell stability, it participates in a variety of essential and dynamic cellular processes including cell migration, cell division, intracellular transport, vesicular trafficking, and organelle morphogenesis. During the past eight years since the green fluorescent protein (GFP) was first used as a marker for the exogenous gene expression, it has been an especially booming era for live cell observations of intracellular movement of many proteins. Because of the dynamic behavior of the cytoskeleton in the cell, GFP has naturally been a vital part of the studies of the cytoskeleton and its associated proteins. In this article, we will describe the advantage of using GFP and how it has been used to study cytoskeletal proteins.