scholarly journals IFT25 is required for the construction of the trypanosome flagellum

2018 ◽  
Author(s):  
Diego Huet ◽  
Thierry Blisnick ◽  
Sylvie Perrot ◽  
Philippe Bastin
Keyword(s):  
Trypanosoma Brucei ◽  
Transport Process ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Bimolecular Fluorescence Complementation ◽  
Live Cells ◽  
Binding Partner ◽  
Eukaryotic Species ◽  
Cilia And Flagella ◽  
The One

AbstractIntraflagellar transport (IFT), the movement of protein complexes responsible for the assembly of cilia and flagella, is remarkably well conserved from protists to humans. However, two IFT components (IFT25 and IFT27) are missing from multiple unrelated eukaryotic species. In mouse, IFT25 and IFT27 are not required for assembly of several cilia with the noticeable exception of the flagellum of spermatozoa. Here we show that the Trypanosoma brucei IFT25 protein is a proper component of the IFT-B complex and displays typical IFT trafficking. Using bimolecular fluorescence complementation assays, we reveal that IFT25 and IFT27 interact within the flagellum in live cells during the IFT transport process. IFT25-depleted cells construct tiny disorganised flagella that accumulate IFT-B proteins (with the exception of IFT27, the binding partner of IFT25) but not IFT-A proteins. This phenotype is comparable to the one following depletion of IFT27 and shows that IFT25/IFT27 constitute a specific module requested for proper IFT and flagellum construction in trypanosomes. We discuss the possible reasons why IFT25/IFT27 would be required for only some types of cilia.

scholarly journals The GTPase IFT27 is involved in both anterograde and retrograde intraflagellar transport

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Diego Huet ◽  
Thierry Blisnick ◽  
Sylvie Perrot ◽  
Philippe Bastin
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Anterograde Transport ◽  
Cargo Transport ◽  
Cilia And Flagella ◽  
Bidirectional Movement

The construction of cilia and flagella depends on intraflagellar transport (IFT), the bidirectional movement of two protein complexes (IFT-A and IFT-B) driven by specific kinesin and dynein motors. IFT-B and kinesin are associated to anterograde transport whereas IFT-A and dynein participate to retrograde transport. Surprisingly, the small GTPase IFT27, a member of the IFT-B complex, turns out to be essential for retrograde cargo transport in Trypanosoma brucei. We reveal that this is due to failure to import both the IFT-A complex and the IFT dynein into the flagellar compartment. To get further molecular insight about the role of IFT27, GDP- or GTP-locked versions were expressed in presence or absence of endogenous IFT27. The GDP-locked version is unable to enter the flagellum and to interact with other IFT-B proteins and its sole expression prevents flagellum formation. These findings demonstrate that a GTPase-competent IFT27 is required for association to the IFT complex and that IFT27 plays a role in the cargo loading of the retrograde transport machinery.

scholarly journals Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes

2008 ◽  
Vol 19 (3) ◽  
pp. 929-944 ◽  
Author(s):  
Sabrina Absalon ◽  
Thierry Blisnick ◽  
Linda Kohl ◽  
Géraldine Toutirais ◽  
Gwénola Doré ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Anterograde Transport ◽  
Body Regions ◽  
Cilia And Flagella ◽  
Bidirectional Movement ◽  
Green Fluorescent

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

scholarly journals The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

2014 ◽  
Vol 25 (17) ◽  
pp. 2620-2633 ◽  
Author(s):  
Thierry Blisnick ◽  
Johanna Buisson ◽  
Sabrina Absalon ◽  
Alexandra Marie ◽  
Nadège Cayet ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
High Concentration ◽  
Anterograde Transport ◽  
Heavy Chains ◽  
Cilia And Flagella ◽  
The Stability ◽  
Intermediate Chains ◽  
Dynein Heavy Chains

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.

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

2020 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Brice Rotureau ◽  
Philippe Bastin
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Live Cell Imaging ◽  
Intraflagellar Transport ◽  
Tsetse Fly ◽  
Tsetse Flies ◽  
Life Cycle Stages ◽  
Summary Statement ◽  
Cilia And Flagella ◽  
Multicellular Organisms

AbstractMulticellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case of Trypanosoma brucei that assembles flagella of 3 to 30 µm during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.Summary statementThis work investigated the assembly of flagella of different length during the development of Trypanosoma brucei in the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.

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

2018 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Cécile Fort ◽  
Thierry Blisnick ◽  
Serge Bonnefoy ◽  
...  
Keyword(s):  
High Resolution ◽  
Focused Ion Beam ◽  
Protein Complexes ◽  
Ion Beam ◽  
Intraflagellar Transport ◽  
Sem Analysis ◽  
Large Individual ◽  
Resolution Electron ◽  
Cilia And Flagella ◽  
Bidirectional Movement

SummaryIntraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. Here 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 occur 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 seconds while remaining on the same side of the axoneme.

scholarly journals A grow-and-lock model for the control of flagellum length in trypanosomes

2018 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Benjamin Morga ◽  
Thierry Blisnick ◽  
Brice Rotureau ◽  
Philippe Bastin
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Daughter Cell ◽  
Linear Growth ◽  
Intraflagellar Transport ◽  
Elongation Rate ◽  
Eukaryotic Cells ◽  
Subsequent Increase ◽  
Normal Length ◽  
Cilia And Flagella

SUMMARYSeveral models have been proposed to explain how eukaryotic cells control the length of their cilia and flagella. Here, we investigated this process in the protistTrypanosoma brucei, an excellent system for cells with stable cilia like photoreceptors or spermatozoa. We show that the total amount of intraflagellar transport material (IFT, the machinery responsible for flagellum construction) increases during flagellum elongation, consistent with constant delivery of precursors and the previously reported linear growth. Reducing the IFT frequency by RNAi knockdown of the IFT kinesin motors slows down the elongation rate and results in the assembly of shorter flagella. These keep on elongating after cell division but fail to reach the normal length. This failure is neither due to an absence of precursors nor to a morphogenetic control by the cell body. We propose that the flagellum is locked after cell division, preventing further elongation or shortening. This is supported by the fact that subsequent increase in the IFT rate does not lead to further elongation. The distal tip FLAM8 protein was identified as a marker for the locking event. It is initiated prior cell division, leading to an arrest of elongation in the daughter cell. Here, we propose a new model termed grow-and-lock where the flagellum elongates until a locking event takes place in a timely defined manner hence fixing length. Alteration in the growth rate and/or in the timing of the locking event would lead to the formation of flagella of different lengths.

scholarly journals Intraflagellar transport—the “new motility” 20 years later

2012 ◽  
Vol 23 (5) ◽  
pp. 751-753 ◽  
Author(s):  
Keith G. Kozminski
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Cellular Mechanisms ◽  
Intracellular Process ◽  
Cilia And Flagella ◽  
Bidirectional Movement

Intraflagellar transport is the rapid, bidirectional movement of protein complexes along the length of most eukaryotic cilia and flagella. Discovery of this intracellular process in Chlamydomonas reinhardtii 20 years ago led to a rapid discovery of cellular mechanisms that underlie a large number of human ciliopathies. Described herein are the events that led to this discovery.

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

2020 ◽  
Vol 133 (18) ◽  
pp. jcs248989
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Brice Rotureau ◽  
Philippe Bastin
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Live Cell Imaging ◽  
Intraflagellar Transport ◽  
Tsetse Fly ◽  
Tsetse Flies ◽  
Life Cycle Stages ◽  
3D Electron Microscopy ◽  
Cilia And Flagella ◽  
Multicellular Organisms

ABSTRACTMulticellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. Trypanosoma brucei assembles flagella of 3 to 30 µm during its development in the tsetse fly. This provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3D electron microscopy and live-cell imaging revealed that IFT was present in all T. brucei life cycle stages. IFT proteins are concentrated at the base, and IFT trains are located along doublets 3–4 and 7–8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of flagellar IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.

scholarly journals Concentration of intraflagellar transport proteins at the ciliary base is required for proper train injection

2021 ◽  
Author(s):  
Jamin Jung ◽  
Julien Santi-Rocca ◽  
Cecile Fort ◽  
Jean-Yves Tinevez ◽  
Cataldo Schietroma ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Live Cell Imaging ◽  
Protein Concentration ◽  
Cell Imaging ◽  
Super Resolution ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Cell Transport ◽  
Cilia And Flagella ◽  
Super Resolution Microscopy

Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.

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

2018 ◽  
Vol 217 (12) ◽  
pp. 4284-4297 ◽  
Author(s):  
Eloïse Bertiaux ◽  
Adeline Mallet ◽  
Cécile Fort ◽  
Thierry Blisnick ◽  
Serge Bonnefoy ◽  
...  
Keyword(s):  
High Resolution ◽  
Focused Ion Beam ◽  
Protein Complexes ◽  
Ion Beam ◽  
Intraflagellar Transport ◽  
Sem Analysis ◽  
Large Individual ◽  
Resolution Electron ◽  
Cilia And Flagella ◽  
Bidirectional Movement

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.

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