high-throughput-gene-tagging-in-trypanosoma-brucei

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

The mechanisms regulating cell layer organisation in developing plant organs are fundamental to plant growth, but remain largely uninvestigated. We have studied the receptor kinase-encoding ARABIDOPSIS CRINKLY4 gene and shown that its expression is restricted to the L1 cell layer of most meristems and organ primordia, including those of the ovule integuments. Insertion mutations show that ARABIDOPSIS CRINKLY4 is required for regulation of cellular organisation during the development of sepal margins and ovule integument outgrowth. We show that ARABIDOPSIS CRINKLY4 encodes a functional kinase that, in ovules and possibly other tissues, is abundant in anticlinal and the inner periclinal plasma membrane of 'outside' cells. We propose that ARABIDOPSIS CRINKLY4 may be involved in maintaining L1 cell layer integrity by receiving and transmitting signals from neighbouring L1 cells and/or from underlying cell layers.

The Arabidopsis ACR4 gene plays a role in cell layer organisation during ovule integument and sepal margin development.

Die Regulierungsmechanismen Zelle Schicht Organisation bei der Entwicklung von Pflanzen Organe sind grundlegend f&uuml;r Pflanzenwachstum, sondern bleiben weitgehend ungekl&auml;rt. Wir haben studierte Rezeptor Kinase-Codierung ARABIDOPSIS CRINKLY4 gen und gezeigt, dass seinen Ausdruck auf die L1-Zellschicht der meisten Meristems und Orgel Primordia, einschlie&szlig;lich derjenigen der Samenanlage Integumente beschr&auml;nkt. Einf&uuml;gung Mutationen zeigen, dass ARABIDOPSIS CRINKLY4 f&uuml;r die Regulierung der zellul&auml;re Organisation w&auml;hrend der Entwicklung von Kelchblatt Margen und Samenanlage Integument Auswuchs erforderlich ist. Wir zeigen, dass ARABIDOPSIS CRINKLY4 eine funktionale Kinase kodiert, die Eizellen und m&ouml;glicherweise anderen Geweben im Antiklinale reichlich vorhanden ist und die inneren periclinal Plasmamembran von &#39;au&szlig;en&#39; Zellen. Wir schlagen ARABIDOPSIS CRINKLY4 Integrit&auml;t L1 Zelle Schicht von senden und empfangen von Signalen aus benachbarten L1 Zellen und/oder zugrunde liegenden Zellschichten beteiligt werden kann.

Das Arabidopsis ACR4-Gen spielt eine Rolle in der Zelle Schicht Organisation w&auml;hrend Samenanlage Integument und Kelchblatt Margenentwicklung.

Trypanosomes evade antibody-mediated lysis via antigenic variation and rapid antibody removal from their cell surface. Recently, in Cell, Engstler et al. (2007) have discovered the mechanism for antibody clearance. Hydrodynamic forces generated by trypanosome swimming create a current, causing surface-bound antibodies to act as "molecular sails." Consequently, they are swept to the cell posterior, internalized via the flagellar-pocket, and degraded. Hydrodynamic sorting is a novel biological process, possibly applicable in other contexts.

Restless gossamers: antibody clearance by hydrodynamic flow forces generated at the surface of motile trypanosome parasites.

Microbial pathogens use environmental cues to trigger the developmental events needed to infect mammalian hosts or transmit to disease vectors. The parasites causing African sleeping sickness respond to citrate or cis-aconitate (CCA) to initiate life-cycle development when transmitted to their tsetse fly vector. This requires hypersensitization of the parasites to CCA by exposure to low temperature, conditions encountered after tsetse fly feeding at dusk or dawn. Here we identify a carboxylate-transporter family, PAD (proteins associated with differentiation), required for perception of this differentiation signal. Consistent with predictions for the response of trypanosomes to CCA, PAD proteins are expressed on the surface of the transmission-competent 'stumpy-form' parasites in the bloodstream, and at least one member is thermoregulated, showing elevated expression and surface access at low temperature. Moreover, RNA-interference-mediated ablation of PAD expression diminishes CCA-induced differentiation and eliminates CCA hypersensitivity under cold-shock conditions. As well as being molecular transducers of the differentiation signal in these parasites, PAD proteins provide the first example of a surface marker able to discriminate the transmission stage of trypanosomes in their mammalian host.

A surface transporter family conveys the trypanosome differentiation signal.

African trypanosomes differentiate between various developmental stages both in mammalian hosts and their tsetse vector to adapt to and survive in the different environments they encounter. In the bloodstream, trypanosomes naturally exist as either proliferative slender-forms or non-proliferative stumpy-forms, the latter being responsible for both prolonged infection and transmission. However, most trypanosome studies are carried out on laboratory-adapted monomorphic cell lines, incapable of differentiating to stumpy-forms or completing the life cycle through the tsetse fly. Partly, this has been due to the inefficiency of transfection of pleomorphic strains which have retained the ability to generate stumpy-forms. Recently, Amaxa Nucleofector® technology was shown to increase transfection efficiency for monomorphic bloodstream forms. Using this technology we have optimised a similar method for pleomorphic bloodstream form transfection, generating transfection efficiencies of 10(-7)-10(-6). This permits routine genetic manipulation of pleomorphic lines, which have the most biological relevance for trypanosomes in the field.

Stable transformation of pleomorphic bloodstream form Trypanosoma brucei.

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F1FO-ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing FO-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F1FO-ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites.

Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes.

One of the first steps in understanding a protein's function is to determine its localization; however, the methods for localizing proteins in some systems have not kept pace with the developments in other fields, creating a bottleneck in the analysis of the large datasets that are generated in the post-genomic era. To address this, we developed tools for tagging proteins in trypanosomatids. We made a plasmid that, when coupled with long primer PCR, can be used to produce transgenes at their endogenous loci encoding proteins tagged at either terminus or within the protein coding sequence. This system can also be used to generate deletion mutants to investigate the function of different protein domains. We show that the length of homology required for successful integration precluded long primer PCR tagging in Leishmania mexicana. Hence, we developed plasmids and a fusion PCR approach to create gene tagging amplicons with sufficiently long homologous regions for targeted integration, suitable for use in trypanosomatids with less efficient homologous recombination than Trypanosoma brucei. Importantly, we have automated the primer design, developed universal PCR conditions and optimized the workflow to make this system reliable, efficient and scalable such that whole genome tagging is now an achievable goal.

A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids.

Plasma membrane-to-plasma membrane connections are common features of eukaryotic cells, with cytoskeletal frameworks below the respective membranes underpinning these connections. A defining feature of Trypanosoma brucei is the lateral attachment of its single flagellum to the cell body, which is mediated by a cytoskeletal structure called the flagellum attachment zone (FAZ). The FAZ is a key morphogenetic structure. Disruption of FAZ assembly can lead to flagellum detachment and dramatic changes in cell shape. To understand this complex structure, the identity of more of its constituent proteins is required. Here, we have used both proteomics and bioinformatics to identify eight new FAZ proteins. Using inducible expression of FAZ proteins tagged with eYFP we demonstrate that the site of FAZ assembly is close to the flagellar pocket at the proximal end of the FAZ. This contrasts with the flagellum, which is assembled at its distal end; hence, these two interconnected cytoskeletal structures have distinct spatially separated assembly sites. This challenging result has many implications for understanding the process of cell morphogenesis and interpreting mutant phenotypes.

Article	Year
The Arabidopsis ACR4 gene plays a role in cell layer organisation during ovule integument and sepal margin development. Development (Cambridge, England)\| PubMed ID: 12900442	2003
Restless gossamers: antibody clearance by hydrodynamic flow forces generated at the surface of motile trypanosome parasites. Cell host & microbe\| PubMed ID: 18005745	2007
A surface transporter family conveys the trypanosome differentiation signal. Nature\| PubMed ID: 19444208	2009
Stable transformation of pleomorphic bloodstream form Trypanosoma brucei. Molecular and biochemical parasitology\| PubMed ID: 23835071	2013
Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23959897	2013
A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open biology\| PubMed ID: 25567099	2015
A dynamic coordination of flagellum and cytoplasmic cytoskeleton assembly specifies cell morphogenesis in trypanosomes. Journal of cell science\| PubMed ID: 25736289	2015

Samuel Dean

Biography

JoVE Journal Publications

Other Publications