Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei.

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper

CEP164C regulates flagellum length in stable flagella

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei . CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella

CEP164C regulates flagellum length in stable flagella

Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella

Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3–4 and 7–8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3–4 s while remaining on the same side of the axoneme

Intraflagellar transport during assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

International audienceSerum osteocalcin was measured in patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism, before or during the treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). Serum osteocalcin and plasma 1,25(OH)2D were decreased in 11 patients with idiopathic hypoparathyroidism before treatment (2.8 +/- 1.27 ng/ml, P less than 0.001 and 14.3 +/- 4.27 pg/ml, P less than 0.001, respectively). In 24 patients with idiopathic hypoparathyroidism during the treatment, serum osteocalcin and plasma 1,25(OH)2D were within the normal range (4.5 +/- 0.74 ng/ml and 25.7 +/- 5.69 pg/ml, respectively). In five patients with pseudohypoparathyroidism before treatment, plasma 1,25(OH)2D was decreased (15.6 +/- 10.6 pg/ml, P less than 0.001) but serum osteocalcin was normal (7.8 +/- 1.66 ng/ml). In nine patients with pseudohypoparathyroidism during the treatment with active vitamin D3, serum osteocalcin and plasma 1,25(OH)2D were normal (6.8 +/- 1.47 ng/ml and 27.2 +/- 6.0 pg/ml, respectively). Serum PTH in pseudohypoparathyroidism was increased before treatment (0.70 +/- 0.34 ng/ml, P less than 0.05) and was normal during the treatment (0.50 +/- 0.13 ng/ml). In idiopathic hypoparathyroidism, the active vitamin D3 increased serum osteocalcin without PTH. In pseudohypoparathyroidism, PTH may increase serum osteocalcin or modulate the effect of active vitamin D3 on serum osteocalcin

SFI1 and centrin form a distal end complex critical for proper centriole architecture and ciliogenesis

Abstract Over the course of evolution, the function of the centrosome has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, as illustrated by the presence of centrioles in humans and a spindle pole body in yeast (SPB). Consistently, the composition of these two core elements has diverged greatly, with the exception of centrin, a protein known to form a complex with Sfi1 in yeast to structurally initiate SPB duplication. Even though SFI1 has been localized to human centrosomes, whether this complex exists at centrioles and whether its function has been conserved is still unclear. Here, using conventional fluorescence and super-resolution microscopies, we demonstrate that human SFI1 is a bona fide centriolar protein localizing to the very distal end of the centriole, where it associates with a pool of distal centrin. We also found that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the specific loss of the distal pool of centrin, without altering centriole duplication in human cells, in contrast to its function for SPB. Instead, we found that SFI1/centrin complexes are essential for correct centriolar architecture as well as for ciliogenesis. We propose that SFI1/centrin complexes may guide centriole growth to ensure centriole integrity and function as a basal body

A key regulatory protein for flagellum length control in stable flagella

Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved

De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form:carbon source preferences and metabolic flux redistributions

Abstract De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway

Metabolic flux distributions in parental and mutant cell lines.

<p>Schemes in panel A compare metabolic flux distribution between the different branches of fatty acid and sterol biosynthesis of the <i>T</i>. <i>brucei</i> procyclic parental and mutant cell lines grown in the carbon source-rich SDM79 medium (<i>in vitro</i>). The carbon sources included in the model are leucine, acetate, glucose and threonine, but not fatty acids, since their incorporation into lipids through <i>de novo</i> biosynthetic pathways has not been demonstrated in rich medium yet. The arrow thickness reflects the strength of metabolic flux redistributions, such as upregulation of leucine metabolism and fatty acid preference, observed in the Δ<i>ivdh</i>, Δ<i>ach</i>/^<i>RNAi</i>ASCT, ^<i>RNAi</i>AceCS, ^<i>RNAi</i>SCP2 and/or ^<i>RNAi</i>TDH/^<i>RNAi</i>PDH mutants compared to the parental PCF cell line. The estimated flux distribution in PCF trypanosomes developing in the tsetse fly midgut is presented in the right box chart. The question mark indicates that the <i>in vivo</i> ketogenic carbon source(s) supplementing threonine, as well as the flux through the acetyl-CoA/HMG-CoA bridge are unknown; this diagram assumes a limited availability of ketogenic carbon sources. Panel B describes metabolic adaptations using as reference the parental PCF grown in rich <i>in vitro</i> conditions. The question mark means that the possible metabolic adaptation <i>in vivo</i> is still unknown, since the carbon source contents in the tsetse's organs, including the gut and salivary glands, remain unknown. In Panel C, these metabolic adaptations are re-interpreted considering the probable physiological conditions that PCF have to face <i>in vivo</i> as reference, with the assumption that ketogenic carbon sources are limited in the tsetse midgut and/or in the salivary glands. Abbreviations: A, acetate; AcCoA, acetyl-CoA; FA, fatty acids; G, glucose; HMGCoA, 3-hydroxy-3-methylglutaryl-CoA; L, leucine; T, threonine; Ste, sterols.</p

Schematic representation of fatty acid and sterol <i>de novo</i> biosynthesis in procyclic trypanosomes.

<p>Black arrows indicate enzymatic steps of leucine, glucose, threonine and acetate metabolism, with dashed arrows symbolizing several steps, to feed fatty acid and ergosterol biosynthesis. Acetyl-CoA and HMG-CoA are boxed to highlight their branching point position. For simplification and clarity only the mitochondrial subcellular compartment is represented. The microsomal elongase system and mitochondrial fatty acid synthesis are represented by a dashed circle labelled ELO and FASII, respectively. The boxed enzymes have been investigated by reverse genetics approaches in this manuscript. Abbreviations: AOB, amino oxobutyrate; Ac-CoA, acetyl-CoA; AcAc-CoA, acetoacetyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA. Indicated enzymes are: ACC, acetyl-CoA carboxylase; ACH, acetyl-CoA thioesterase; AceCS, AMP-dependent acetyl-CoA synthetase; AKCT, 2-amino-3-ketobutyrate CoA transferase; ASCT, acetate:succinate CoA-transferase; BCAT, branched-chain aminotransferase; BCKDH, branched-chain α-keto acid dehydrogenase complex; HMGR, HMG-CoA reductase; HMGS, HMG-CoA synthase; IVDH, isovaleryl-CoA dehydrogenase; MCC, 3-methylcrotonoyl-CoA decarboxylase; MGH, 3-methylglutaconyl-CoA hydratase; PDH, pyruvate dehydrogenase complex; SCP2, SCP2-thiolase; TDH, threonine 3-dehydrogenase.</p