scholarly journals The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ahmad Abdelzaher Zaki Khalifa ◽  
Muneyoshi Ichikawa ◽  
Daniel Dai ◽  
Shintaroh Kubo ◽  
Corbin Steven Black ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tetrahymena Thermophila ◽  
Building Blocks ◽  
Post Translational Modifications ◽  
Ciliary Beating ◽  
Cryo Electron Microscopy ◽  
The Stability ◽  
And Function ◽  
Β Tubulin ◽  
Junction Proteins

Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

scholarly journals The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

2019 ◽  
Author(s):  
Ahmad Khalifa ◽  
Muneyoshi Ichikawa ◽  
Daniel Dai ◽  
Shintaroh Kubo ◽  
Corbin Black ◽  
...  
Keyword(s):  
Building Blocks ◽  
Post Translational Modifications ◽  
Ciliary Beating ◽  
Structural Support ◽  
Cryo Electron Microscopy ◽  
The Common ◽  
Species Specific ◽  
And Function ◽  
Standing Question ◽  
Junction Proteins

AbstractMicrotubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the α- and β-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.Using cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

Post-translational modifications and stabilization of microtubules regulate transport of viral factors during infections

2021 ◽  
Vol 49 (4) ◽  
pp. 1735-1748
Author(s):  
Silvia Requena ◽  
Francisco Sánchez-Madrid ◽  
Noa B. Martín-Cófreces
Keyword(s):  
Immune Responses ◽  
Immune Cells ◽  
Building Blocks ◽  
Cell Types ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Associated Proteins ◽  
The Stability ◽  
Different Characteristics ◽  
Β Tubulin

Tubulin post-translational modifications (PTMs) constitute a source of diversity for microtubule (MT) functions, in addition to the different isotypes of α and β-tubulin acting as building blocks of MTs. Also, MT-associated proteins (MAPs) confer different characteristics to MTs. The combination of all these factors regulates the stability of these structures that act as rails to transport organelles within the cell, facilitating the association of motor complexes. All these functions are involved in crucial cellular processes in most cell types, ranging from spindle formation in mitosis to the defense against incoming cellular threats during phagocytosis mediated by immune cells. The regulation of MT dynamics through tubulin PTMs has evolved to depend on many different factors that act in a complex orchestrated manner. These tightly regulated processes are particularly relevant during the induction of effective immune responses against pathogens. Viruses have proved not only to hijack MTs and MAPs in order to favor an efficient infection, but also to induce certain PTMs that improve their cellular spread and lead to secondary consequences of viral processes. In this review, we offer a perspective on relevant MT-related elements exploited by viruses.

scholarly journals Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers

2021 ◽  
Vol 22 (11) ◽  
pp. 6000
Author(s):  
Sara Bertuzzi ◽  
Ana Gimeno ◽  
Ane Martinez-Castillo ◽  
Marta G. Lete ◽  
Sandra Delgado ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Lectin Binding ◽  
Building Blocks ◽  
Cryo Electron Microscopy ◽  
Hpma Copolymers ◽  
Hydroxypropyl Methacrylamide ◽  
Galectin 3 ◽  
Nmr Methods ◽  
The Individual ◽  
Statistical Effects

The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.

scholarly journals cAMP-stimulated phosphorylation of diaphanous 1 regulates protein stability and interaction with binding partners in adrenocortical cells

2013 ◽  
Vol 24 (6) ◽  
pp. 848-857 ◽  
Author(s):  
Donghui Li ◽  
Eric B. Dammer ◽  
Natasha C. Lucki ◽  
Marion B. Sewer
Keyword(s):  
Oxysterol Binding Protein ◽  
Mitochondrial Movement ◽  
Binding Partners ◽  
Extracellular Signal ◽  
Camp Signaling Pathway ◽  
Integral Role ◽  
Mitochondrial Trafficking ◽  
The Stability ◽  
And Function ◽  
Β Tubulin

Diaphanous homologue 1 (DIAPH1) is a Rho effector protein that coordinates cellular dynamics by regulating microfilament and microtubule function. We previously showed that DIAPH1 plays an integral role in regulating the production of cortisol by controlling the rate of mitochondrial movement, by which activation of the adrenocorticotropin (ACTH)/cAMP signaling pathway stimulates mitochondrial trafficking and promotes the interaction between RhoA and DIAPH1. In the present study we use mass spectrometry to identify DIAPH1 binding partners and find that DIAPH1 interacts with several proteins, including RhoA, dynamin-1, kinesin, β-tubulin, β-actin, oxysterol-binding protein (OSBP)–related protein 2 (ORP2), and ORP10. Moreover, DIAPH1 is phosphorylated in response to dibutyryl cAMP (Bt2cAMP) at Thr-759 via a pathway that requires extracellular signal-related kinase (ERK). Alanine substitution of Thr-759 renders DIAPH1 more stable and attenuates the interaction between DIAPH1 and kinesin, ORP2, and actin but has no effect on the ability of the protein to interact with RhoA or β-tubulin. Finally, overexpression of a DIAPH1 T759A mutant significantly decreases the rate of Bt2cAMP-stimulated mitochondrial movement. Taken together, our findings establish a key role for phosphorylation in regulating the stability and function of DIAPH1.

scholarly journals The structure of the yeast Ctf3 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Stephen M Hinshaw ◽  
Andrew N Dates ◽  
Stephen C Harrison
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
High Resolution ◽  
Atomic Model ◽  
Kinetochore Assembly ◽  
Cryo Electron Microscopy ◽  
Molecular Interpretation ◽  
Spindle Microtubules ◽  
And Function ◽  
Assembly Site

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

scholarly journals Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

2021 ◽  
Author(s):  
Philipp Milkereit ◽  
Gisela Pöll ◽  
Michael Pilsl ◽  
Joachim Griesenbeck ◽  
Herbert Tschochner
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Protein Assembly ◽  
Functional Coupling ◽  
Large Ribosomal Subunit ◽  
Cryo Electron Microscopy ◽  
Intrinsic Quality ◽  
Domain Arrangement ◽  
The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

scholarly journals The physiological butyrylcholinesterase tetramer is a dimer of dimers stabilized by a superhelical assembly

2018 ◽  
Author(s):  
Miguel Ricardo Leung ◽  
Laura S. van Bezouwen ◽  
Lawrence M. Schopfer ◽  
Joel L. Sussman ◽  
Israel Silman ◽  
...  
Keyword(s):  
Practical Importance ◽  
Residence Times ◽  
Catalytic Domains ◽  
Polyproline Ii ◽  
Cryo Electron Microscopy ◽  
Tetramerization Domain ◽  
Polyproline Ii Helix ◽  
Quaternary Structures ◽  
The Stability ◽  
And Function

AbstractThe quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localisation and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryo-electron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 Å. Our structure reveals that the BChE tetramer is organised as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central anti-parallel polyproline II helix (PRAD). The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the HuBChE tetramer. Our cryo-EM structure reveals the basis for assembly of the physiological tetramers, and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases, and may provide a promising template for designing other proteins with improved circulatory residence times.

scholarly journals The structure of the yeast Ctf3 complex

2019 ◽  
Author(s):  
Stephen M. Hinshaw ◽  
Andrew N. Dates ◽  
Stephen C. Harrison
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
High Resolution ◽  
Atomic Model ◽  
Kinetochore Assembly ◽  
Cryo Electron Microscopy ◽  
Molecular Interpretation ◽  
Spindle Microtubules ◽  
And Function ◽  
Assembly Site

ABSTRACTKinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture. We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

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