Indicator species characterization and removal in a detention pond in the Plaster Creek watershed.

Microbial pathogen contamination is a leading cause of impairment for urban rivers and streams in Michigan. Reports on the ability of green infrastructure best management practices to remove microbial pathogens have been highly variable. This study evaluated the influence of a detention basin (Kreiser Pond) on microbial dynamics in the Plaster Creek watershed in West Michigan. High levels of fecal indicator bacteria and coliphage were documented in influent and effluent water, with significant increases in indicator microbe concentrations during storm events. In dry conditions, Kreiser Pond efficiently reduced the number of indicator microbes flowing through the basin. Rainfall volume had a greater influence on the diversity of bacteria than sampling location. Antibiotic resistance was prevalent in culturable E. coli from Kreiser Pond, demonstrating a potential public health risk and highlighting the need for identifying the ultimate sources of microbial pollution.

NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.

A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways.

Early endosomal cargo is typically targeted to either a degradative or recycling pathway. Despite established functions for the retromer and ESCRT complexes at late endosomes/multivesicular bodies, the mechanisms integrating and coordinating these functions remain largely unknown. Rab family GTPases are key membrane trafficking organizers and could contribute. Here, in the unicellular organism Trypanosoma brucei, we demonstrate that Rab28 locates to the endosomal pathway and partially colocalizes with Vps23, an ESCRT I component. Rab28 is required for turnover of endocytosed proteins and for lysosomal delivery of protein cargo. Using RNA interference we find that in Rab28-depleted cells, protein levels of ESCRT I (Vps23/28) and retromer (Vps26) are also decreased, suggesting that Rab28 is an important regulator of these factors. We suggest that Rab28 coordinates the activity of retromer-dependent trafficking and ESCRT-mediated degradative pathways.

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

Rab11 function in Trypanosoma brucei: identification of conserved and novel interaction partners.

The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei, where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens, interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

Evidence for a shared nuclear pore complex architecture that is conserved from the last common eukaryotic ancestor.

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.

A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

A novel Rho-like protein TbRHP is involved in spindle formation and mitosis in trypanosomes.

BACKGROUND: In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.10.6240). Further, only a single RhoGAP-like protein (Tb09.160.4180) is annotated, contrasting with the >70 Rho GAP proteins from Homo sapiens. We wished to establish the function(s) of TbRHP and if Tb09.160.4180 is a potential GAP for this protein. METHODS/FINDINGS: TbRHP represents an evolutionarily restricted member of the Rho GTPase clade and is likely trypanosomatid restricted. TbRHP is expressed in both mammalian and insect dwelling stages of T. brucei and presents with a diffuse cytoplasmic location and is excluded from the nucleus. RNAi ablation of TbRHP results in major cell cycle defects and accumulation of multi-nucleated cells, coinciding with a loss of detectable mitotic spindles. Using yeast two hybrid analysis we find that TbRHP interacts with both Tb11.01.3180 (TbRACK), a homolog of Rho-kinase, and the sole trypanosome RhoGAP protein Tb09.160.4180, which is related to human OCRL. CONCLUSIONS: Despite minimization of the Rho pathway, TbRHP retains an important role in spindle formation, and hence mitosis, in trypanosomes. TbRHP is a partner for TbRACK and an OCRL-related trypanosome Rho-GAP.

Identification of the major cysteine protease of Giardia and its role in encystation.

Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.