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 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 Advances in High-Speed Structured Illumination Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Tianyu Zhao ◽  
Zhaojun Wang ◽  
Tongsheng Chen ◽  
Ming Lei ◽  
Baoli Yao ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Light Power ◽  
Recent Developments ◽  
Super Resolution Microscopy

Super-resolution microscopy surpasses the diffraction limit to enable the observation of the fine details in sub-cellular structures and their dynamics in diverse biological processes within living cells. Structured illumination microscopy (SIM) uses a relatively low illumination light power compared with other super-resolution microscopies and has great potential to meet the demands of live-cell imaging. However, the imaging acquisition and reconstruction speeds limit its further applications. In this article, recent developments all targeted at improving the overall speed of SIM are reviewed. These comprise both hardware and software improvements, which include a reduction in the number of raw images, GPU acceleration, deep learning and the spatial domain reconstruction. We also discuss the application of these developments in live-cell imaging.

Expansion Microscopy: Scalable and Convenient Super-Resolution Microscopy

2019 ◽  
Vol 35 (1) ◽  
pp. 683-701 ◽  
Author(s):  
Paul W. Tillberg ◽  
Fei Chen
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Resolving Power ◽  
Relative Ease ◽  
Nanoscale Structures ◽  
Physical Form ◽  
Tissue Specimens ◽  
Super Resolution Microscopy

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick.

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 Stochastic models of polymerization based axonal actin transport

2019 ◽  
Author(s):  
Nilaj Chakrabarty ◽  
Peter Jung
Keyword(s):  
Molecular Basis ◽  
Transport Mechanism ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Length Distribution ◽  
Super Resolution ◽  
Slow Axonal Transport ◽  
Simple Diffusion ◽  
Nucleation Sites ◽  
Super Resolution Microscopy

AbstractPulse-chase and radio-labeling studies have shown that actin is transported in bulk along the axon at rates consistent with slow axonal transport. In a recent paper, using a combination of live cell imaging, super resolution microscopy and computational modeling, we proposed that biased polymerization of metastable actin fibers (actin trails) along the axon shaft forms the molecular basis of bulk actin transport. The proposed mechanism is unusual, and can be best described as molecular hitch hiking, where G-actin molecules are intermittently incorporated into actin fibers which grow preferably in anterograde direction giving rise to directed transport, released after the fibers collapse only to be incorporated into another fiber. In this paper, we use our computational model to make additional predictions that can be tested experimentally to further scrutinize our proposed mechanism for bulk actin transport. In the previous paper the caliber of our model axon, the density of the actin nucleation sites to form the metastable actin fibers, the length distribution of the actin trails and their growth rate were adapted to the biologic axons used for measurements. Here we predict how the transport rate will change with axon caliber, density of nucleation sites, nucleation rates and trail lengths. We also discuss why a simple diffusion-based transport mechanism can not explain bulk actin transport.

scholarly journals Super-resolution Microscopy Approaches for Live Cell Imaging

2014 ◽  
Vol 107 (8) ◽  
pp. 1777-1784 ◽  
Author(s):  
Antoine G. Godin ◽  
Brahim Lounis ◽  
Laurent Cognet
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Super Resolution Microscopy

scholarly journals Stochastic association of neighboring replicons creates replication factories in budding yeast

2013 ◽  
Vol 202 (7) ◽  
pp. 1001-1012 ◽  
Author(s):  
Nazan Saner ◽  
Jens Karschau ◽  
Toyoaki Natsume ◽  
Marek Gierliński ◽  
Renata Retkute ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Super Resolution ◽  
Replication Proteins ◽  
Replication Factory ◽  
Genome Wide ◽  
Nuclear Structures ◽  
Super Resolution Microscopy ◽  
Insight Into

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

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