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.

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 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—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 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 Cryo Focused Ion Beam Applications in High Resolution Electron Microscopy Studies of Beam Sensitive Crystals

2019 ◽  
Vol 25 (S2) ◽  
pp. 1402-1403 ◽  
Author(s):  
Daliang Zhang ◽  
Nini Wei ◽  
Lingmei Liu ◽  
Kepeng Song ◽  
Ali Behzad ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
High Resolution Electron Microscopy ◽  
Resolution Electron

3D electron microscopy investigations of human dentin at the micro/nano-scale using focused ion beam based nanostructuring

RSC Advances ◽  
2015 ◽  
Vol 5 (10) ◽  
pp. 7196-7199 ◽  
Author(s):  
Meltem Sezen ◽  
Sina Sadighikia
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
High Resolution Electron Microscopy ◽  
High Definition ◽  
Transmission Electron ◽  
Resolution Electron ◽  
Human Dentin ◽  
Microscopy Techniques

In this study, high resolution electron microscopy techniques, such as Focused Ion Beam (FIB), Scanning Electron Microscopy (SEM) and High Resolution Transmission Electron Microscopy (HRTEM) allowed for revealing micro/nano features within human dentin with high definition and accuracy.

FIB Enhancement of Mechanically Polished Cross-sections

2019 ◽  
Author(s):  
Becky Holdford
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Cross Sections ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
High Resolution Imaging ◽  
Chemical Attack ◽  
Resolution Imaging ◽  
Scanning Electron

Abstract On mechanically polished cross-sections, getting a surface adequate for high-resolution imaging is sometimes beyond the analyst’s ability, due to material smearing, chipping, polishing media chemical attack, etc.. A method has been developed to enable the focused ion beam (FIB) to re-face the section block and achieve a surface that can be imaged at high resolution in the scanning electron microscope (SEM).

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