Microtubule specialization by +TIP networks: from mechanisms to functional implications.

To fulfill their actual cellular role, individual microtubules become functionally specialized through a broad range of mechanisms. The 'search and capture' model posits that microtubule dynamics and functions are specified by cellular targets that they capture (i.e., a posteriori), independently of the microtubule-organizing center (MTOC) they emerge from. However, work in budding yeast indicates that MTOCs may impart a functional identity to the microtubules they nucleate, a priori. Key effectors in this process are microtubule plus-end tracking proteins (+TIPs), which track microtubule tips to regulate their dynamics and facilitate their targeted interactions. In this review, we discuss potential mechanisms of a priori microtubule specialization, focusing on recent findings indicating that +TIP networks may undergo liquid biomolecular condensation in different cell types.

Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent.

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

Control of microtubule organization and dynamics: two ends in the limelight.

Microtubules have fundamental roles in many essential biological processes, including cell division and intracellular transport. They assemble and disassemble from their two ends, denoted the plus end and the minus end. Significant advances have been made in our understanding of microtubule plus-end-tracking proteins (+TIPs) such as end-binding protein 1 (EB1), XMAP215, selected kinesins and dynein. By contrast, information on microtubule minus-end-targeting proteins (-TIPs), such as the calmodulin-regulated spectrin-associated proteins (CAMSAPs) and Patronin, has only recently started to emerge. Here, we review our current knowledge of factors, including microtubule-targeting agents, that associate with microtubule ends to control the dynamics and function of microtubules during the cell cycle and development.

Structural basis of the 9-fold symmetry of centrioles.

The centriole, and the related basal body, is an ancient organelle characterized by a universal 9-fold radial symmetry and is critical for generating cilia, flagella, and centrosomes. The mechanisms directing centriole formation are incompletely understood and represent a fundamental open question in biology. Here, we demonstrate that the centriolar protein SAS-6 forms rod-shaped homodimers that interact through their N-terminal domains to form oligomers. We establish that such oligomerization is essential for centriole formation in C. elegans and human cells. We further generate a structural model of the related protein Bld12p from C. reinhardtii, in which nine homodimers assemble into a ring from which nine coiled-coil rods radiate outward. Moreover, we demonstrate that recombinant Bld12p self-assembles into structures akin to the central hub of the cartwheel, which serves as a scaffold for centriole formation. Overall, our findings establish a structural basis for the universal 9-fold symmetry of centrioles.

Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.

An EB1-binding motif acts as a microtubule tip localization signal.

Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.

Recruitment dynamics of juvenile salmonids: Comparisons among populations and with classic case studies.

Understanding recruitment, the process by which individuals are added to a population or to a fishery, is critical for understanding population dynamics and facilitating sustainable fisheries management. Important variation in recruitment dynamics is observed among populations, wherein some populations exhibit asymptotic productivity and others exhibit overcompensation (i.e., compensatory density-dependence in recruitment). Our ability to understand this interpopulation variability in recruitment patterns is limited by a poor understanding of the underlying mechanisms, such as the complex interactions between density dependence, recruitment, and environment. Furthermore, most studies on recruitment are conducted using an observational design with long time series that are seldom replicated across populations in an experimentally controlled fashion. Without proper replication, extrapolations between populations are tenuous, and the underlying environmental trends are challenging to quantify. To address these issues, we conducted a field experiment manipulating stocking densities of juvenile brook trout Salvelinus fontinalis in three wild populations to show that these neighboring populations-which exhibit divergent patterns of density dependence due to environmental conditions-also have important differences in recruitment dynamics. Testing against four stock-recruitment models (density independent, linear, Beverton-Holt, and Ricker), populations exhibited ~twofold variation in asymptotic productivity, with no overcompensation following a Beverton-Holt model. Although environmental variables (e.g., temperature, pH, depth, substrate) correlated with population differences in recruitment, they did not improve the predictive power in individual populations. Comparing our patterns of recruitment with classic salmonid case studies revealed that despite differences in the shape and parameters of the curves (i.e., Ricker vs. Beverton-Holt), a maximum stocking density of about five YOY fish/m2 emerged. Higher densities resulted in very marginal increases in recruitment (Beverton-Holt) or reduced recruitment due to overcompensation (Ricker).

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action.

In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-to-straight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns.

Microtubule minus-end regulation at a glance.

Microtubules are cytoskeletal filaments essential for numerous aspects of cell physiology. They are polarized polymeric tubes with a fast growing plus end and a slow growing minus end. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge on the dynamics and organization of microtubule minus ends. Several factors, including the γ-tubulin ring complex, CAMSAP/Patronin, ASPM/Asp, SPIRAL2 (in plants) and the KANSL complex recognize microtubule minus ends and regulate their nucleation, stability and interactions with partners, such as microtubule severing enzymes, microtubule depolymerases and protein scaffolds. Together with minus-end-directed motors, these microtubule minus-end targeting proteins (-TIPs) also control the formation of microtubule-organizing centers, such as centrosomes and spindle poles, and mediate microtubule attachment to cellular membrane structures, including the cell cortex, Golgi complex and the cell nucleus. Structural and functional studies are starting to reveal the molecular mechanisms by which dynamic -TIP networks control microtubule minus ends.

Taxanes convert regions of perturbed microtubule growth into rescue sites.

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method.

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.

The importance of skin manifestations, serology and nailfold (video)capillaroscopy in morphea and systemic sclerosis: current understanding and new insights.

Since the field around morphea and systemic sclerosis (SSc) is evolving rapidly, this review approaches conventional as well as more recent clinical developments from a dermatological point of view. Skin manifestations are critical in sub-classifying these diseases ensuring a correct prognosis for these patients. They can be discretely present, and therefore, diagnosis can be challenging sometimes, implicating a thorough dermatological examination is mandatory. Furthermore, a growing amount of dermatologists perform nailfold videocapillaroscopy (NVC), a more recent reliable non-invasive imaging technique used for in vivo assessment of the microcirculation at the nailfold. After all, specific NVC-changes are present in a majority of patients with SSc. This way, dermatologists not only take part in the diagnosis process through clinical investigation but also through the use of a modern state of the art imaging technique that is becoming the golden standard in SSc multidisciplinary workup. In this review, current understandings for NVC in morphea and SSc are revised. So far, the role of NVC in the diagnosis/prognosis/classification of morphea patients has not been thoroughly investigated to make proper conclusions. As for SSc, it is well known that NVC contributes to the diagnosis and can make a fundamental difference especially when obvious clinical SSc signs are absent. This review emphasizes the (somewhat underestimated) role of dermatologists in the process of diagnosis and follow-up, and thus, the difference we can make for our patients and fellow colleagues in the multidisciplinary workup of SSc and morphea.

Exploitation of Tolerance of Wheat Kernel Weight and Shape-Related Traits from Aegilops tauschii under Heat and Combined Heat-Drought Stresses.

Kernel weight and shape-related traits are inherited stably and increase wheat yield. Narrow genetic diversity limits the progress of wheat breeding. Here, we evaluated kernel weight and shape-related traits and applied genome-wide association analysis to a panel of wheat multiple synthetic derivative (MSD) lines. The MSD lines harbored genomic fragments from Aegilops tauschii. These materials were grown under optimum conditions in Japan, as well as under heat and combined heat-drought conditions in Sudan. We aimed to explore useful QTLs for kernel weight and shape-related traits under stress conditions. These can be useful for enhancing yield under stress conditions. MSD lines possessed remarkable genetic variation for all traits under all conditions, and some lines showed better performance than the background parent Norin 61. We identified 82 marker trait associations (MTAs) under the three conditions; most of them originated from the D genome. All of the favorable alleles originated from Ae. tauschii. For the first time, we identified markers on chromosome 5D associated with a candidate gene encoding a RING-type E3 ubiquitin-protein ligase and expected to have a role in regulating wheat seed size. Our study provides important knowledge for the improvement of wheat yield under optimum and stress conditions. The results emphasize the importance of Ae. tauschii as a gene reservoir for wheat breeding.

[ISO 1999:2013 part 1 : Revised probability model for calculating noise-induced hearing loss]. / ISO 1999:2013 Teil 1 : Überarbeitetes Wahrscheinlichkeitsmodell zur Berechnung des lärmbedingten Hörverlusts.

The ISO 1999:2013 norm describes a method of calculating the statistically expected permanent threshold shift (PTS) due to noise. Input parameters are noise level LEX,8h related to an 8­hour working day, duration of noise exposure in years, gender, and age. The background is a formula based on four datasets of measured values from larger surveys. Within its defined scope, ISO 1999 provides audiometric hearing curves for the frequencies 0.5, 1, 2, 3, 4, and 6 kHz for probability percentiles 5 to 95. This international standard is a useful additional tool for estimating the most probable cause of hearing disability when compared to the hearing curve in a noise worker's threshold audiogram and thus for deciding whether an occupational noise-induced hearing loss is likely to be present or not. According to the formula given in ISO 1999, sets of curves were recalculated separately for women and men to make them easily accessible in a new, expanded, and modernized graphical representation in this publication. Thus, according to ISO 1999, the following applies for the assessment: The higher the age, the more likely a noise-induced hearing threshold shift is to recede behind an age-related hearing threshold shift.

Comprehensive Analysis of Binding Sites in Tubulin.

Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin-protein and tubulin-ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation.

Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence.

The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC50; 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to ß-tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.

Tracking the ends: a dynamic protein network controls the fate of microtubule tips.

Microtubule plus-end tracking proteins (+TIPs) are a diverse group of evolutionarily conserved cellular factors that accumulate at the ends of growing microtubules. They form dynamic networks through the interaction of a limited set of protein modules, repeat sequences and linear motifs that bind to each other with moderate affinities. +TIPs regulate different aspects of cell architecture by controlling microtubule dynamics, microtubule interactions with cellular structures and signalling factors, and the forces that are exerted on microtubule networks.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.