Theodore R. Muth

Identification of Sorting Determinants in the C-terminal Cytoplasmic Tails of the γ-Aminobutyric Acid Transporters GAT-2 and GAT-3

In order to perform their physiologic functions, polarized epithelial cells must target ion transport proteins to the appropriate domains of their plasma membranes. Molecular signals responsible for polarized sorting have been identified for several membrane proteins which span the bilayer once. Most ion transport proteins are polytopic, however, and little is known of the signals responsible for the targeting of this class of polypeptides. Members of the γ-aminobutyric acid (GABA) transporter family are polytopic membrane proteins found endogenously in both epithelial cells and neurons. We have identified narrowly defined sequences which are required for the proper accumulation of two members of this transporter family in Madin-Darby canine kidney cells. The highly homologous GABA transporter isoforms, GAT-2 and GAT-3, localize to the basolateral and apical surfaces, respectively, when expressed stably in Madin-Darby canine kidney cells. We have generated deletion constructs and chimeric transporters composed of complimentary portions of GAT-2 and GAT-3. We find that information which directs their differential sorting is present in the C-terminal cytoplasmic tails of these two polypeptides. A sequence of 22 amino acids at the C terminus of GAT-2 is required for the transporter’s basolateral distribution and is capable of directing GAT-3 to the basolateral surface when appended to the C terminus of this normally apical polypeptide. The deletion of 32 amino acids from the C terminus of GAT-3 causes this transporter to become mislocalized to both surfaces. Moreover, removal of the final three amino acids of GAT-3 (THF) similarly disrupts its apical sorting. The GAT-3 C-terminal sequence resembles motifs which interact with PDZ domains, raising the possibility that the steady state distribution of GAT-3 at the apical plasmalemmal surface requires a protein-protein interaction mediated by its extreme C-terminal cytoplasmic tail. These data provide the first characterization of a protein-based signal required for the apical distribution of a membrane protein.

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Localization of the effector-specifying regions of Gi2alpha and Gqalpha.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of α subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of α subunit-effector specificity, we localized the effector-specifying regions of αi2, which inhibits adenylyl cyclase, and αq, which stimulates phosphoinositide phospholipase C using chimeric α subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both αi2 and αq were localized within the GTPase domain. An αq/αi2/αq chimera containing only 78 αi2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This αi2 segment includes regions corresponding to two of the three regions of αs that activate adenylyl cyclase, but does not include any of the α subunit regions that switch conformation upon binding GTP. Replacement of the αq residues that comprise the helical domain with the homologous αi2 residues resulted in a chimeric α subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of αs and αt, our results suggest that the effector specificity of α subunits is generally determined by the GTPase and not the helical domain.

Auranofin and related heterometallic gold(I)–thiolates as potent inhibitors of methicillin-resistant Staphylococcus aureus bacterial strains

A series of new heterometallic gold(I) thiolates containing ferrocenyl-phoshines were synthesized. Their antimicrobial properties were studied and compared to that of FDA-approved drug, auranofin (Ridaura), prescribed for the treatment of rheumatoid arthritis. MIC in the order of one digit micromolar were found for most of the compounds against Gram-positive bacteria Staphylococcus aureus and CA MRSA strains US300 and US400. Remarkably, auranofin inhibited S. aureus, US300 and US400 in the order of 150-300 nM. This is the first time that the potent inhibitory effect of auranofin on MRSA strains has been described. The effects of a selected heterometallic compound and auranofin were also studied in a non-tumorigenic human embryonic kidney cell line (HEK-293).

Chapter 4 Protein Trafficking in Polarized Cells

Epithelial cells line the lumens of organs and thus constitute the interface between the bodys interior and exterior surfaces. This position endows these cells with the important task of regulating what enters and what is exported from the body. In order to accomplish this function, epithelia must have structurally and functionally distinct membrane surfaces: the apical surface exposed to the lumen, and the basolateral surface in contact with the laterally adjacent epithelial cells, and the connective tissue and capillary network below the epithelia. The specific lipid and protein contents of the apical and basolateral membrane surfaces are determined by a number of sorting and retention mechanisms. Many of these sorting and retention mechanisms are shared with other polarized cell types including neurons and certain cells of the immune system. This chapter focuses on recent advances in understanding how these various mechanisms facilitate the generation, maintenance, and dynamic regulation of protein and lipid trafficking within epithelial cells.

Sorting of ion pumps in polarized epithelial cells.

The physiologic functions of a P-type ATPase are determined not only by its catalytic and regulatory properties but also by its distribution among a cells various membranous compartments. With polarized epithelial cells that mediate vectorial ion fluxes, the restriction of P-type ion pumps to one or the other distinct domains of the plasmalemma in large measure determines the parent tissues solute and fluid transport capacities. The biologic significance of these anisotropic distributions is well illustrated by the mechanisms through which the Na,K-ATPase drives the majority of active epithelial secretory and absorptive processes.] In most epithelial cells, the Na,K-ATPase is restricted to the basolateral plasmalemmal domain.2 This membrane surface rests on the epithelial basement membrane, is in contact with the extracellular fluid compartment, and is involved in extensive contacts with neighboring epithelial cells. The basolateral membrane is separated by tight junctions from the apical plasmalemma, which generally confronts a compartment that is topologically continuous with the extracorporeal space. The nonequilibrium ion distributions generated by the sodium pump are exploited by secondary active transport systems to drive uphill secretory and absorptive fluxes. By expressing different classes of transport systems and restricting their distributions to one or the other surface compartment, epithelial cells can use the basolateral population of the Na,K-ATPase to catalyze a remarkably diverse array of unidirectional transport processes. To achieve polarized distribution of P-type ATPase proteins, epithelial cells must be able to target newly synthesized ion pumps to the correct membrane surfaces and to retain them there following their delivery. To participate in these sorting functions, P-type ATPase subunit polypeptides must encode information within their structures that specify their sites of ultimate functional residence. Furthermore, machinery within the epithelial cell must be able to recognize this information and act on its messages3 Efforts to understand the nature of these sorting signals and of the cellular components that interpret them have largely relied on the extensive homology that relates the members of the P-type ATPase family. This high degree of structural

Sorting and trafficking of ion transport proteins in polarized epithelial cells.

Transport proteins are targeted to specific plasmalemmal domains in polarized epithelial cells. The molecular signals that govern these sorting events are just beginning to be elucidated. In many cases, the cell surface delivery of transport proteins is subjected to tight regulation. Several different mechanisms appear to participate in these trafficking processes.

Characterizing urban soils in New York City: profile properties and bacterial communities

PurposeThe influence of human activities on the development and functioning of urban soils and their profile characteristics is still inadequately understood. Microbial communities can change due to anthropogenic disturbances and it is unclear how they exist along urban soil profiles. This study investigates the dynamic soil properties (DSPs) and the bacterial communities along the profiles of urban soils in New York City (NYC) with varying degree of human disturbances.Materials and methodsEleven pedons were investigated across NYC as well as one control soil in a nearby non-urban area. Six soils are formed in naturally deposited materials (ND) and five in human-altered and human-transported materials (HAHT). For each soil, the profile was described and each horizon was sampled to assess DSPs and the bacterial community composition and diversity.Results and discussionThe development and the DSPs of NYC soils are influenced by the incorporation of HAHT materials and atmospheric deposits. The most abundant bacterial taxa observed in the NYC soils are also present in most natural and urban soils worldwide. The bacterial diversity was lower in some soils formed in ND materials, in which the contribution of low-abundance taxa was more restricted. Some differences in bacterial community composition separated the soils formed in ND materials and in dredged sediments from the soils formed in high artifact fill and serpentinite till. Changes in bacterial community composition between soil horizons were more noticeable in urban soils formed in ND materials than in those formed in HAHT materials which display less differentiated profiles and in the non-urban highly weathered soil.ConclusionsThe bacterial diversity is not linked to the degree of disturbance of the urban soils but the variations in community composition between pedons and along soil profiles could be the result of changes in soil development and properties related to human activities and should be consistently characterized in urban soils.

EXPRESSION OF NEUROTRANSMITTER TRANSPORT SYSTEMS IN POLARIZED CELLS

Publisher Summary This chapter describes the expression of neurotransmitter transport systems in polarized cells. Members of the neurotransmitter transport protein family are normally expressed in polarized cells. The chapter examines the mechanisms through which they attain their specific subcellular distributions. Cultured epithelial cell lines can be readily transfected with complementary DNAs (cDNAs) encoding transporter proteins. When expressed in this setting, the neurotransmitter transport proteins are targeted to specific subdomains of the plasma membrane. Their distributions can be evaluated by immunocytochemical, biochemical, and transport assays. Transporters can also be transiently expressed in neurons in culture. They appear to be segregated to the specific subcellular structures, which correlate with their predicted functions. The chapter also presents an analysis of exogenous protein expression by fluorescence microscopy.

Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots

Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 104 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 105 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots.