The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L . mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L . mexicana mutants in vivo . In mixed infections of the permissive sand fly vector Lutzomyia longipalpis , paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L . mexicana need directional motility for successful colonisation of sand flies.
Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate.
23The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during 24 the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a 25 short immotile flagellum in amastigotes residing in mammalian phagocytes. This study 26 examined the protein composition and in vivo function of the promastigote flagellum. Protein 27 mass spectrometry and label free protein enrichment testing of isolated flagella and 28 deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms 29 2 (available via ProteomeXchange with identifier PXD011057). This information was used to 30 generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This 31 first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered 32 swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster 33 swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including 34 aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar 35 rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode 36 sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in 37 vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed 38 promastigotes and uncoordinated swimmers were severely diminished in the fly after 39 defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant 40 lacking the central pair protein PF16 showed that these paralysed promastigotes did not 41 reach anterior regions of the fly alimentary tract. These data show that L. mexicana need 42 directional motility for successful colonisation of sand flies. 43 Author Summary 44Leishmania are protozoan parasites, transmitted between mammals by the bite of 45 phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is 46 motile and used for anchoring the parasites to prevent clearance with the digested blood 47 meal remnants. To dissect flagellar functions and their importance in life cycle progression, 48we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-49Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro 50 before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant 51 swimming speeds showed that about half behaved differently compared to the wild type: a 52 few swam faster, many slower and some were completely paralysed. We also found a group 53 of uncoordinated swimmers. To test whether flagellar motility is required for parasite 54 migration from the fly midgut to the foregut from where they reach the next host, we infected 55 sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking 56 these tags up to nine days after infection showed that paralysed and uncoordinated 57 Leishmania were rapidly lost from flies. ...
The human mediator subunit MED25 acts as a coactivator that binds the transcriptional activation domains (TADs) present in various cellular and viral gene-specific transcription factors. Previous studies, including on NMR measurements and site-directed mutagenesis, have only yielded low-resolution models that are difficult to refine further by experimental means. Here, we apply computational molecular dynamics simulations to study the interactions of two different TADs from the human transcription factor ETV5 (ERM) and herpes virus VP16-H1 with MED25. Like other well-studied coactivator-TAD complexes, the interactions of these intrinsically disordered domains with the coactivator surface are temporary and highly dynamic (‘fuzzy’). Due to the fact that the MED25 TAD-binding region is organized as an elongated cleft, we specifically asked whether these TADs are capable of binding in either orientation and how this could be achieved structurally and energetically. The binding of both the ETV5 and VP16-TADs in either orientation appears to be possible but occurs in a conformationally distinct manner and utilizes different sets of hydrophobic residues present in the TADs to drive the interactions. We propose that MED25 and at least a subset of human TADs specifically evolved a redundant set of molecular interaction patterns to allow binding to particular coactivators without major prior spatial constraints.
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