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.

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

3D printing alias additive manufacturing can transform 3D virtual models created by computer-aided design (CAD) into physical 3D objects in a layer-by-layer manner dispensing with conventional molding or machining. Since the incipiency, significant advancements have been achieved in understanding the process of 3D printing and the relationship of component, structure, property and application of the created objects. Because hydrogels are one of the most feasible classes of ink materials for 3D printing and this field has been rapidly advancing, this Review focuses on hydrogel designs and development of advanced hydrogel-based biomaterial inks and bioinks for 3D printing. It covers 3D printing techniques including laser printing (stereolithography, two-photon polymerization), extrusion printing (3D plotting, direct ink writing), inkjet printing, 3D bioprinting, 4D printing and 4D bioprinting. It provides a comprehensive overview and discussion of the tailorability of material, mechanical, physical, chemical and biological properties of hydrogels to enable advanced hydrogel designs for 3D printing. The range of hydrogel-forming polymers covered encompasses biopolymers, synthetic polymers, polymer blends, nanocomposites, functional polymers, and cell-laden systems. The representative biomedical applications selected demonstrate how hydrogel-based 3D printing is being exploited in tissue engineering, regenerative medicine, cancer research, in vitro disease modeling, high-throughput drug screening, surgical preparation, soft robotics and flexible wearable electronics. Incomparable by thermoplastics, thermosets, ceramics and metals, hydrogel-based 3D printing is playing a pivotal role in the design and creation of advanced functional (bio)systems in a customizable way. An outlook on future directions of hydrogel-based 3D printing is presented.

3D printing of hydrogels: Rational design strategies and emerging biomedical applications

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the 'tubulin code'. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

/pdf/the-tubulin-code-and-its-role-in-controlling-microtubule-13ooa1q7rl.pdf

The tubulin code and its role in controlling microtubule properties and functions.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

/pdf/nuclear-pores-as-versatile-reference-standards-for-4jdyoeuza3.pdf

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

https://hal.archives-ouvertes.fr/hal-02391963/document

Microtubule-Associated Proteins: Structuring the Cytoskeleton.

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

/pdf/imaging-cellular-ultrastructures-using-expansion-microscopy-5fquvpym46.pdf

Imaging cellular ultrastructures using expansion microscopy (U-ExM).

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.U-ExM enables near-native expansion microscopy of samples in vitro and in cells. The combination of U-ExM with confocal microscopy and HyVolution revealed details of centriole chirality that were previously accessible only by electron microscopy.

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.

https://science.sciencemag.org/content/sci/363/6424/285.full.pdf

Marketa Schmidt-Cernohorska

Papers

Imaging cellular ultrastructures using expansion microscopy (U-ExM).

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

Flagellar microtubule doublet assembly in vitro reveals a regulatory role of tubulin C-terminal tails.