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Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures. We determine the microtubule structure across all three species at between 3.0 and 3.6 Å resolution by cryo-electron microscopy and find small differences at the β-tubulin lateral interactions. Using in vitro reconstitution assays and quantitative biochemistry, we show that tubulin's free energy scales inversely with temperature. The observed weakening of lateral contacts and the low apparent activation energy for tubulin incorporation provide an explanation for the overall stability and higher growth rates of microtubules in cold-adapted frog species. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Eukaryotic cells typically express multiple tubulin isoforms that form the microtubule cytoskeleton. A new study of the evolution and functional diversification of pools of tubulin isoforms suggests that these proteins are part of a co-evolving network that includes the extensive microtubule interactome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets. 1-5 While microtubules and their molecular building block αβ-tubulin are established drug targets in a variety of cancers, 6 , 7 we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and β-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications. The protocol is applicable to any cell or tissue of interest. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1-3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5-8], the contribution of the spindle's main building block, the αβ-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics in vitro. X. laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X. tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our in vitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Fused in sarcoma (FUS) is an RNA binding protein involved in regulating many aspects of RNA processing and linked to several neurodegenerative diseases. Transcriptomics studies indicate that FUS binds a large variety of RNA motifs, suggesting that FUS RNA binding might be quite complex. Here, we present solution structures of FUS zinc finger (ZnF) and RNA recognition motif (RRM) domains bound to RNA. These structures show a bipartite binding mode of FUS comprising of sequence-specific recognition of a NGGU motif via the ZnF and an unusual shape recognition of a stem-loop RNA via the RRM. In addition, sequence-independent interactions via the RGG repeats significantly increase binding affinity and promote destabilization of structured RNA conformation, enabling additional binding. We further show that disruption of the RRM and ZnF domains abolishes FUS function in splicing. Altogether, our results rationalize why deciphering the RNA binding mode of FUS has been so challenging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The primarily nuclear RNA-binding protein FUS (fused in sarcoma) forms pathological cytoplasmic inclusions in a subset of early-onset amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. In response to cellular stress, FUS is recruited to cytoplasmic stress granules, which are hypothesized to act as precursors of pathological inclusions. We monitored the stress-induced nucleocytoplasmic shuttling of endogenous FUS in an ex vivo mouse CNS model and human neural networks. We found that hyperosmolar, but not oxidative, stress induced robust cytoplasmic translocation of neuronal FUS, with transient nuclear clearance and loss of function. Surprisingly, this reaction is independent of stress granule formation and the molecular pathways activated by hyperosmolarity. Instead, it represents a mechanism mediated by cytoplasmic redistribution of Transportin 1/2 and is potentiated by transcriptional inhibition. Importantly, astrocytes, which remain unaffected in ALS/FTD-FUS, are spared from this stress reaction that may signify the initial event in the development of FUS pathology., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although the parts list is nearly complete for many cellular structures, mechanisms that control their size remain poorly understood. Loughlin and colleagues now show that phosphorylation of a single residue of katanin, a microtubule-severing protein, largely accounts for the difference in spindle length between two closely related frogs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.