Tetrahymena RIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

ABSTRACTDoublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged Microtubule Inner Proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical and imaging techniques we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins co-localize to Tetrahymena cilia and basal bodies, but assemble independently. Cryo-electron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4 and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.SUMMARYMicrotubule Inner Proteins (MIPs) bind to the luminal surface of highly stable microtubules. Combining cell biology and cryo-electron tomography, Stoddard et al. show that RIB72A and RIB72B are conserved MIPs in ciliary doublet microtubules and that they are important for proper ciliary motility.

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Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0405 ◽

2018 ◽

Vol 29 (21) ◽

pp. 2566-2577 ◽

Cited By ~ 20

Author(s):

Daniel Stoddard ◽

Ying Zhao ◽

Brian A. Bayless ◽

Long Gui ◽

Panagiota Louka ◽

...

Keyword(s):

High Speed ◽

Biological Function ◽

Imaging Techniques ◽

Protein Composition ◽

Structural Defects ◽

Power Stroke ◽

Basal Bodies ◽

Structural Assembly ◽

Stable Core ◽

Cilia And Flagella

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic microtubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.

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Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity

Life Science Alliance ◽

10.26508/lsa.202101185 ◽

2021 ◽

Vol 5 (3) ◽

pp. e202101185

Author(s):

Irene Riera-Tur ◽

Tillman Schäfer ◽

Daniel Hornburg ◽

Archana Mishra ◽

Miguel da Silva Padilha ◽

...

Keyword(s):

Protein Aggregation ◽

Protein Trafficking ◽

Cell Biology ◽

Electron Tomography ◽

Lysosomal Storage ◽

Gain Of Function ◽

Cryo Electron Tomography ◽

A Subunit ◽

Late Stages ◽

Β Sheet

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like β-sheet proteins (β proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, β proteins interact with and sequester AP-3 μ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3μ1 expression ameliorates neurotoxicity caused by β proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

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Structural insights into flagellar stator–rotor interactions

eLife ◽

10.7554/elife.48979 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 14

Author(s):

Yunjie Chang ◽

Ki Hwan Moon ◽

Xiaowei Zhao ◽

Steven J Norris ◽

MD A Motaleb ◽

...

Keyword(s):

Conformational Change ◽

High Speed ◽

Electron Tomography ◽

Deletion Mutants ◽

Flagellar Motor ◽

Wild Type ◽

Cryo Electron Tomography ◽

Flagellar Filament ◽

Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e19-01-0063 ◽

2019 ◽

Vol 30 (15) ◽

pp. 1805-1816 ◽

Cited By ~ 14

Author(s):

Erin E. Dymek ◽

Jianfeng Lin ◽

Gang Fu ◽

Mary E. Porter ◽

Daniela Nicastro ◽

...

Keyword(s):

Cell Motility ◽

Electron Tomography ◽

Structural Studies ◽

Ciliary Beating ◽

Ciliary Motility ◽

Functional Studies ◽

Cryo Electron Tomography ◽

Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

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Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

10.1101/684316 ◽

2019 ◽

Author(s):

Long Gui ◽

Kangkang Song ◽

Douglas Tristchler ◽

Raqual Bower ◽

Yan Si ◽

...

Keyword(s):

Electron Tomography ◽

Core Complex ◽

Ciliary Motility ◽

C Terminus ◽

Large N ◽

Cryo Electron Tomography ◽

N Terminus ◽

Regulatory Complex ◽

Cilia And Flagella ◽

Linker Domain

ABSTRACTThe nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits” associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.Significance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1–11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

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The molecular architecture of engulfment during Bacillus subtilis sporulation

eLife ◽

10.7554/elife.45257 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Bacillus Subtilis ◽

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

High Spatial Resolution ◽

Electron Tomography ◽

Ion Beam ◽

Molecular Architecture ◽

Native State ◽

Cryo Electron Tomography

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

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Electrically tunable lenses – eliminating mechanical axial movements during high-speed 3D live imaging

Journal of Cell Science ◽

10.1242/jcs.258650 ◽

2021 ◽

Vol 134 (16) ◽

Author(s):

Christoforos Efstathiou ◽

Viji M. Draviam

Keyword(s):

Cell Biology ◽

High Speed ◽

Imaging Techniques ◽

Three Dimensional ◽

Cost Effective ◽

High Speed Imaging ◽

Liquid Lenses ◽

Live Cell Microscopy ◽

Tunable Lenses ◽

Cell Microscopy

ABSTRACT The successful investigation of photosensitive and dynamic biological events, such as those in a proliferating tissue or a dividing cell, requires non-intervening high-speed imaging techniques. Electrically tunable lenses (ETLs) are liquid lenses possessing shape-changing capabilities that enable rapid axial shifts of the focal plane, in turn achieving acquisition speeds within the millisecond regime. These human-eye-inspired liquid lenses can enable fast focusing and have been applied in a variety of cell biology studies. Here, we review the history, opportunities and challenges underpinning the use of cost-effective high-speed ETLs. Although other, more expensive solutions for three-dimensional imaging in the millisecond regime are available, ETLs continue to be a powerful, yet inexpensive, contender for live-cell microscopy.

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The molecular architecture of engulfment during Bacillussubtilis sporulation

10.1101/498220 ◽

2018 ◽

Author(s):

Kanika Khanna ◽

Javier Lopez-Garrido ◽

Ziyi Zhao ◽

Reika Watanabe ◽

Yuan Yuan ◽

...

Keyword(s):

Mother Cell ◽

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Molecular Model ◽

Ion Beam ◽

Leading Edge ◽

Cellular Structures ◽

Molecular Architecture ◽

Cryo Electron Tomography

AbstractThe study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation inBacillus subtilisusing cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.

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Fully automated, sequential focused ion beam milling for cryo-electron tomography

10.1101/797514 ◽

2019 ◽

Author(s):

Tobias Zachs ◽

João M. Medeiros ◽

Andreas Schertel ◽

Gregor L. Weiss ◽

Jannik Hugener ◽

...

Keyword(s):

Cell Biology ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Model Organisms ◽

User Input ◽

Cryo Electron Tomography ◽

Cellular Context ◽

Rough Milling ◽

Time Required

AbstractCryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, with the unique ability to determine structures of macromolecular complexes in their cellular context. A major limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

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