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 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 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 Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides

2008 ◽  
Vol 19 (1) ◽  
pp. 274-283 ◽  
Author(s):  
Rosemarie V. Barkus ◽  
Olga Klyachko ◽  
Dai Horiuchi ◽  
Barry J. Dickson ◽  
William M. Saxton
Keyword(s):  
Transport Mechanism ◽  
Fluorescent Protein ◽  
Retrograde Transport ◽  
Major Contributor ◽  
Anterograde Transport ◽  
Microtubule Motor ◽  
Fast Transport ◽  
Green Fluorescent ◽  
Neuronal Functions ◽  
Axonal Swellings

A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.

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 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.

Cytoplasmic dynein-associated structures move bidirectionallyin vivo

2002 ◽  
Vol 115 (7) ◽  
pp. 1453-1460 ◽  
Author(s):  
Shuo Ma ◽  
Rex L. Chisholm
Keyword(s):  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Anterograde Transport ◽  
Directional Movement ◽  
Motor Activities ◽  
Direction Of Movement ◽  
Linear Movement ◽  
Bidirectional Movement ◽  
Green Fluorescent

Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubulebased motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively,different motors could associate with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse their direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo association but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end-directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.

Intraflagellar transport motors in Caenorhabditis elegans neurons

2004 ◽  
Vol 32 (5) ◽  
pp. 682-684 ◽  
Author(s):  
J.M. Scholey ◽  
G. Ou ◽  
J. Snow ◽  
A. Gunnarson
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Protein Fusion ◽  
C Elegans ◽  
Sensory Cilia ◽  
Macromolecular Protein ◽  
Green Fluorescent

IFT (intraflagellar transport) assembles and maintains sensory cilia on the dendritic endings of chemosensory neurons within the nematode Caenorhabditis elegans. During IFT, macromolecular protein complexes called IFT particles (which carry ciliary precursors) are moved from the base of the sensory cilium to its distal tip by anterograde IFT motors (kinesin-II and Osm-3 kinesin) and back to the base by retrograde IFT-dynein [Rosenbaum and Witman (2002) Nat. Rev. Mol. Cell Biol. 3, 813–825; Scholey (2003) Annu. Rev. Cell Dev. Biol. 19, 423–443; and Snell, Pan and Wang (2004) Cell 117, 693–697]. In the present study, we describe the protein machinery of IFT in C. elegans, which we have analysed using time-lapse fluorescence microscopy of green fluorescent protein-fusion proteins in concert with ciliary mutants.

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 Electron-tomographic analysis of intraflagellar transport particle trains in situ

2009 ◽  
Vol 187 (1) ◽  
pp. 135-148 ◽  
Author(s):  
Gaia Pigino ◽  
Stefan Geimer ◽  
Salvatore Lanzavecchia ◽  
Eugenio Paccagnini ◽  
Francesca Cantele ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Sensory Function ◽  
Anterograde Transport ◽  
Transmission Electron ◽  
Transport Particle ◽  
Cilia And Flagella ◽  
Bidirectional Movement ◽  
The Individual ◽  
Tomographic Analysis

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

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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