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 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 Dynamics of the IFT machinery at the ciliary tip

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Alexander Chien ◽  
Sheng Min Shih ◽  
Raqual Bower ◽  
Douglas Tritschler ◽  
Mary E Porter ◽  
...  
Keyword(s):  
Basal Body ◽  
Full Range ◽  
Intraflagellar Transport ◽  
Feedback Mechanism ◽  
Conventional Imaging ◽  
Range Of Movement ◽  
Anterograde Transport ◽  
Traffic Jam ◽  
Negative Feedback Mechanism ◽  
Cilia And Flagella

Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

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.

Correlative Nanomechanical Measurements for Complex Engineered Systems

MRS Advances ◽  
2016 ◽  
Vol 1 (12) ◽  
pp. 799-804 ◽  
Author(s):  
Eric D. Hintsala ◽  
Syed Asif ◽  
Douglas D. Stauffer
Keyword(s):  
Contact Pressure ◽  
Ex Situ ◽  
Metallic Oxide ◽  
Complex Engineered Systems ◽  
Transmission Electron ◽  
Important Input ◽  
Engineered Systems ◽  
The Individual ◽  
Disc Drive

ABSTRACTMultilayered film stacks, with length scales less than 10 nm are commonly used in a variety of devices, but present significant challenges to mechanical testing and evaluation. This is due to property convolution of the different layers. Both the properties of the individual layers and the combined response of the film stack are important input for design optimization. Here, we present ex-situ nanoindentation of a film stack representative of a perpendicular magnetic recording (PMR) hard disc drive (HDD), with more than 10 layers. We then compare this with in-situ transmission electron microscopy indentation to visualize deformation of individual layers of the stack. The ex-situ testing reveals early plastic deformation, with an initially high contact pressure (13 GPa) and modulus ( >160 GPa), followed by significant softening (8 GPa contact pressure and 140 GPa modulus), then slight hardening to 9 GPa. From in-situ testing, it is revealed that the metallic layer directly under the diamond like carbon (DLC) contributes the majority of the deformation and plastic flow, which is in turn constrained by a metallic oxide.

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.

Epitaxial Growth and Stability of C49 TiSi2 ON Si(111).

1990 ◽  
Vol 198 ◽  
Author(s):  
Hyeongtag Jeon ◽  
J. W. Honeycutt ◽  
C. A. Sukow ◽  
T. P. Humphreys ◽  
R. J. Nemanich ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Diffraction ◽  
Metastable Phase ◽  
Plan View ◽  
Transmission Electron ◽  
Temperature Deposition ◽  
The Individual ◽  
Relationship Of

ABSTRACTEpitaxial TiSi2 films have been grown by molecular beam epitaxy (MBE) on atomically clean Si(111)-orientated substrates. The growth procedure involves the ambient temperature deposition of Ti films of 50Å thickness and annealing to 800°C. In situ low energy electron diffraction (LEED) and Auger electron spectroscopy (AES) techniques have been used to monitor the TiSi2 formation process. The epitaxial films have been identified as the C49 metastable phase by both Raman spectroscopy and electron diffraction. Plan view transmission electron microscopy shows three different connected island morphologies. The individual island structures are single crystal and are grown epitaxially with different crystallographic orientations. The orientational relationship of the largest islands is given by [3 1 1] C49 TiSi2//[112]Si and (130) C49 TiSi2//(l1 1)Si. High resolution transmission electron microscopy (HRTEM) cross-section shows a coherent interface extending over several hundred angstroms.

Amorphisation and Recrystallisation of Nanometre Sized Zones in Silicon

2005 ◽  
Vol 108-109 ◽  
pp. 145-150 ◽  
Author(s):  
P.D. Edmondson ◽  
S.E. Donnelly ◽  
R.C. Birtcher
Keyword(s):  
Electron Microscopy ◽  
Amorphous Material ◽  
Heavy Ion ◽  
Initial Size ◽  
Transmission Electron ◽  
Wide Range ◽  
Ion Impact ◽  
Amorphous Zone ◽  
The Individual

In this paper we present a detailed study in which the formation, by heavy ion impact, and thermal recrystallisation of individual amorphous zones have been studied using in-situ transmission electron microscopy. In agreement with previous work, we observe a reduction in the total volume of amorphous material contained within the amorphous zones following thermal annealing over a wide range of temperatures. When the evolution of the individual amorphous zones is followed, those with similar starting sizes are observed to recrystallise over a range of temperatures from 70 °C to 500 °C. The temperature at which an amorphous zone fully recrystallises does not appear to be correlated with initial size. In addition, zones are occasionally observed to increase in size temporarily on some isochronal annealing steps. Furthermore, observations during a ramp anneal show that many zones recrystallise in a stepwise manner separated by periods of stability. These phenomenon are discussed in terms of the I-V pair.

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.

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