scholarly journals Microtubule plus-end regulation by centriolar cap proteins

2021 ◽  
Author(s):  
Funso E. Ogunmolu ◽  
Shoeib Moradi ◽  
Vladimir A. Volkov ◽  
Chris van Hoorn ◽  
Jingchao Wu ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Electron Tomography ◽  
In Vitro Reconstitution ◽  
Luminal Side ◽  
Cryo Electron Tomography ◽  
Direct Binding ◽  
Distal Centriole

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS-4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus-end dynamics and centriole biogenesis.

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

HIV-1 Biogenesis Studied by Cellular Cryo-Electron Tomography and Biochemical in vitro Reconstitution

2012 ◽  
Vol 18 (S2) ◽  
pp. 50-51
Author(s):  
L. Carlson ◽  
J.H. Hurley ◽  
K. Grünewald ◽  
H. Kräusslich ◽  
J. Briggs
Keyword(s):  
Electron Tomography ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Hiv 1

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

scholarly journals Structural insight into TPX2-stimulated microtubule assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Rui Zhang ◽  
Johanna Roostalu ◽  
Thomas Surrey ◽  
Eva Nogales
Keyword(s):  
Molecular Mechanism ◽  
Structural Information ◽  
Microtubule Assembly ◽  
Cryo Electron Microscopy ◽  
Interaction Mode ◽  
In Vitro Reconstitution ◽  
Assembly Factor ◽  
Ran Gtp ◽  
Mitosis And Meiosis

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

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

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
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.

scholarly journals Targeted dynein inhibition generates flagellar beating

2017 ◽  
Author(s):  
Jianfeng Lin ◽  
Daniela Nicastro
Keyword(s):  
Molecular Mechanism ◽  
Structural Changes ◽  
Direct Evidence ◽  
Electron Tomography ◽  
Asymmetric Distribution ◽  
Conformational Switching ◽  
Cryo Electron Tomography ◽  
Cilia And Flagella ◽  
Biochemical Analyses ◽  
Sea Urchin Sperm

Motile cilia and flagella are highly conserved organelles that are essential for the normal development and health of many eukaryotes including humans. To reveal the molecular mechanism of motility, we used cryo-electron tomography of active sea urchin sperm flagella to directly visualize the macromolecular complexes and their structural changes during flagellar beating. We resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. Our results provide direct evidence for the conformational switching predicted by the ‘switch-point-hypothesis’. However, they also reveal a fundamentally different mechanism of generating motility by inhibiting dyneins, rather than activating them, causing an asymmetric distribution of force and thus bending. Our high-resolution structural and biochemical analyses provide a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating in an all-or-none manner.

scholarly journals Current data processing strategies for cryo-electron tomography and subtomogram averaging

2021 ◽  
Vol 478 (10) ◽  
pp. 1827-1845
Author(s):  
Euan Pyle ◽  
Giulia Zanetti
Keyword(s):  
Data Processing ◽  
Electron Tomography ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Current Data ◽  
Processing Strategies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
The Individual

Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets.

scholarly journals In situ architecture of neuronal α-Synuclein inclusions

2020 ◽  
Author(s):  
Victoria A. Trinkaus ◽  
Irene Riera-Tur ◽  
Antonio Martínez-Sánchez ◽  
Felix J.B. Bäuerlein ◽  
Qiang Guo ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Electron Tomography ◽  
Structural Flexibility ◽  
Multiple Systems ◽  
Cryo Electron Tomography ◽  
Multiple Systems Atrophy ◽  
The Brain ◽  
Cellular Organelles

Summaryα-Synuclein (α-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinson’s disease (PD) and multiple systems atrophy (MSA)1,2. α-Syn aggregates spread throughout the brain during disease progression2, suggesting mechanisms of intercellular seeding. Formation of α-Syn amyloid fibrils is observed in vitro3,4 and fibrillar α-Syn has been purified from patient brains5,6, but recent reports questioned whether disease-relevant α-Syn aggregates are fibrillar in structure7-9. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like α-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of α-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived α-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by α-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal α-Syn inclusions contain α-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.

scholarly journals Topological Insights into Mutant Huntingtin Exon 1 and PolyQ Aggregates by Cryo-Electron Tomography

2020 ◽  
Author(s):  
Jesús G. Galaz-Montoya ◽  
Sarah H. Shahmoradian ◽  
Koning Shen ◽  
Judith Frydman ◽  
Wah Chiu
Keyword(s):  
Trinucleotide Repeat ◽  
Electron Tomography ◽  
Mutant Huntingtin ◽  
And Topology ◽  
Cryo Electron Tomography ◽  
Exon 1 ◽  
Subtomogram Averaging ◽  
The Brain ◽  
Polyq Tract

ABSTRACTHuntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we observed some filaments in both types of aggregates under ∼2 nm in width, thinner than previously reported, while other regions form large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy, slab-shaped morphology in both aggregates, supportive of the “polyQ core” model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling and their identification in cells without fusion tags.

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