The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human ...β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.
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•TUBB3 point mutations near the kinesin-binding site change polymerization dynamics•Mutations alter dynamic instability parameters and end-stabilizing cap lifetimes•TUBB3 point mutation alters the distribution of tubulin conformations in solution•3.8-Å cryo-EM structures of microtubules with wild-type and mutant human TUBB3
The organization of diverse microtubule-based structures depends on regulated tubulin polymerization and directional transport. Ti et al. show that mutations of residues on the microtubule lattice surface and proximal to the kinesin-binding site, but distal from the GTP-hydrolysis site and inter-subunit contacts, alter polymerization dynamics at both microtubule ends.
Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may ...directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo-electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.
Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting ...our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo‐focused ion beam milling‐enabled cryo‐electron tomography to image sperm flagella from three mammalian species. We resolve in‐cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament‐bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament‐bracing structures reinforcing microtubules at the nano‐scale to accessory structures that impose micron‐scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
SYNOPSIS
Diverse organisms and cell types rely on evolutionarily ancient and structurally conserved motile cilia to swim through a broad range of fluid environments. Here, in situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Mammalian sperm flagella are reinforced by micron‐scale accessory structures and nano‐scale microtubule inner proteins.
The connecting piece at the base of the flagellum forms an intricate, asymmetric chamber around the sperm centrioles.
The atypical distal centriole is composed of doublet microtubules splayed out around two singlet microtubules.
Outer dense fibers are directly coupled to axonemal doublet microtubules in the principal piece, but not in the midpiece.
In situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function ...relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.
Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro ...from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end-tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.
The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on ...microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ∼10-20 enzyme-labeled complexes in 100 μl of sample (∼10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome
. It governs the synthesis, translocation, membrane ...insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation
, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.
Nature provides much inspiration for the design of multistep conversion processes, with numerous reactions running simultaneously and without interference in cells, for example. A key challenge in ...mimicking nature's strategies is to compartmentalize incompatible reagents and catalysts, for example, for tandem catalysis. Here, we present a new strategy for antagonistic catalyst compartmentalization. The synthesis of bifunctional Janus catalyst particles carrying acid and base groups on the particle's opposite patches is reported as is their application as acid‐base catalysts in oil/water emulsions. The synthesis strategy involved the use of monodisperse, hydrophobic and amine‐functionalized silica particles (SiO2−NH2−OSi(CH3)3) to prepare an oil‐in‐water Pickering emulsion (PE) with molten paraffin wax. After solidification, the exposed patch of the silica particles was selectively etched and refunctionalized with acid groups to yield acid‐base Janus particles (Janus A–B). These materials were successfully applied in biphasic Pickering interfacial catalysis for the tandem dehydration‐Knoevenagel condensation of fructose to 5‐(hydroxymethyl)furfural‐2‐diethylmalonate (5‐HMF‐DEM) in a water/4‐propylguaiacol PE. The results demonstrate the advantage of rapid extraction of 5‐hydroxymethylfurfural (5‐HMF), a prominent platform molecule prone to side product formation in acidic media. A simple strategy to tune the acid/base balance using PE with both Janus A–B and monofunctional SiO2−NH2−OSi(CH3)3 base catalysts proved effective for antagonistic tandem catalysis.
Hopes and spheres: A new strategy is presented for antagonistic catalyst compartmentalization by immobilizing acid and base groups onto the opposite patches of Janus spheres using a solid wax synthesis method. The bifunctional catalytic emulsifiers are successfully employed in biphasic oil/water emulsions for interfacial catalysis of a dehydration‐Knoevenagel condensation.
Danger patterns on microbes or damaged host cells bind and activate C1, inducing innate immune responses and clearance through the complement cascade. How these patterns trigger complement initiation ...remains elusive. Here, we present cryo-electron microscopy analyses of C1 bound to monoclonal antibodies in which we observed heterogeneous structures of single and clustered C1-immunoglobulin G1 (IgG1) hexamer complexes. Distinct C1q binding sites are observed on the two Fc-CH2 domains of each IgG molecule. These are consistent with known interactions and also reveal additional interactions, which are supported by functional IgG1-mutant analysis. Upon antibody binding, the C1q arms condense, inducing rearrangements of the C1r
s
proteases and tilting C1q's cone-shaped stalk. The data suggest that C1r may activate C1s within single, strained C1 complexes or between neighboring C1 complexes on surfaces.
Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information ...defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.
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