Chapter 4 The Kinesin-3 Family

The Kinesin-3s are a family of cargo transporters. They typically display highly processive plus-end-directed motion, either as dimers or in teams, formed via interaction with carg

Measuring microtubule dynamics

Microtubules are key players in cellular self-organization, acting as structural scaffolds, cellular highways, force generators and signalling platforms. Microtubules are polar filaments that undergo dynamic instability, i.e. transition between phases of growth and shrinkage. This allows microtubules to explore the inner space of the cell, generate pushing and pulling forces and remodel themselves into arrays with different geometry and function such as the mitotic spindle. To do this, eukaryotic cells employ an arsenal of regulatory proteins to control microtubule dynamics spatially and temporally. Plants and microorganisms have developed secondary metabolites that perturb microtubule dynamics, many of which are in active use as cancer chemotherapeutics and anti-inflammatory drugs. Here we summarise the methods used to visualise microtubules and to measure the parameters of dynamic instability to study both microtubule regulatory proteins and the action of small molecules interfering with microtubule assembly and/or disassembly

Repurposing screen identifies mebendazole as a clinical candidate to synergise with docetaxel for prostate cancer treatment

BACKGROUND: Docetaxel chemotherapy in prostate cancer has a modest impact on survival. To date, efforts to develop combination therapies have not translated into new treatments. We sought to develop a novel therapeutic strategy to tackle chemoresistant prostate cancer by enhancing the efficacy of docetaxel. METHODS: We performed a drug-repurposing screen by using murine-derived prostate cancer cell lines driven by clinically relevant genotypes. Cells were treated with docetaxel alone, or in combination with drugs (n = 857) from repurposing libraries, with cytotoxicity quantified using High Content Imaging Analysis. RESULTS: Mebendazole (an anthelmintic drug that inhibits microtubule assembly) was selected as the lead drug and shown to potently synergise docetaxel-mediated cell killing in vitro and in vivo. Dual targeting of the microtubule structure was associated with increased G2/M mitotic block and enhanced cell death. Strikingly, following combined docetaxel and mebendazole treatment, no cells divided correctly, forming multipolar spindles that resulted in aneuploid daughter cells. Liposomes entrapping docetaxel and mebendazole suppressed in vivo prostate tumour growth and extended progression-free survival. CONCLUSIONS: Docetaxel and mebendazole target distinct aspects of the microtubule dynamics, leading to increased apoptosis and reduced tumour growth. Our data support a new concept of combined mebendazole/docetaxel treatment that warrants further clinical evaluation

Spatial positioning of EB family proteins at microtubule tips involves distinct nucleotide-dependent binding properties

EB proteins track the ends of growing microtubules and regulate microtubule dynamics both directly and by acting as the hub of the tip-tracking network. Mammalian cells express cell type-specific combinations of three EB proteins with different cellular roles. Here we reconstitute EB1, EB2 and EB3 tip tracking in vitro. We find that all three EBs show rapid exchange at the microtubule tip and that their signal correlates to the microtubule assembly rate. However, the three signals differ in their maxima and the position from the microtubule tip. Using microtubules built with nucleotide analogues and site-directed mutagenesis, we show that EB2 prefers binding to microtubule lattices containing a 1:1 mixture of different nucleotides and its distinct binding specificity is conferred by amino acid substitutions at the right-hand side interface of the EB microtubule-binding domain with tubulin. Our data are consistent with the model that all three EB paralogs sense the nucleotide state of both β-tubulins flanking their binding site. Their different profile of preferred binding sites contributes to occupying spatially distinct domains at the temporally evolving microtubule tip structure

Patients with chronic pain exhibit individually unique cortical signatures of pain encoding

Chronic pain is characterised by an ongoing and fluctuating intensity over time. Here, we investigated how the trajectory of the patients\u27 endogenous pain is encoded in the brain. In repeated functional MRI (fMRI) sessions, 20 patients with chronic back pain and 20 patients with chronic migraine were asked to continuously rate the intensity of their endogenous pain. Linear mixed effects models were used to disentangle cortical processes related to pain intensity and to pain intensity changes. At group level, we found that the intensity of pain in patients with chronic back pain is encoded in the anterior insular cortex, the frontal operculum, and the pons; the change of pain in chronic back pain and chronic migraine patients is mainly encoded in the anterior insular cortex. At the individual level, we identified a more complex picture where each patient exhibited their own signature of endogenous pain encoding. The diversity of the individual cortical signatures of chronic pain encoding results bridge between clinical observations and neuroimaging; they add to the understanding of chronic pain as a complex and multifaceted disease

Placing taxonomists at the heart of a definitive and comprehensive global resource on the world's plants

It is time to synthesize the knowledge that has been generated through more than 260 years of botanical exploration, taxonomic and, more recently, phylogenetic research throughout the world. The adoption of an updated Global Strategy for Plant Conservation (GSPC) in 2011 provided the essential impetus for the development of the World Flora Online (WFO) project. The project represents an international, coordinated effort by the botanical community to achieve GSPC Target 1, an electronic Flora of all plants. It will be a first‐ever unique and authoritative global source of information on the world's plant diversity, compiled, curated, moderated and updated by an expert and specialist‐based community (Taxonomic Expert Networks – “TENs” – covering a taxonomic group such as family or order) and actively managed by those who have compiled and contributed the data it includes. Full credit and acknowledgement will be given to the original sources, allowing users to refer back to the primary data. A strength of the project is that it is led and endorsed by a global consortium of more than 40 leading botanical institutions worldwide. A first milestone for producing the World Flora Online is to be accomplished by the end of 2020, but the WFO Consortium is committed to continuing the WFO programme beyond 2020 when it will develop its full impact as the authoritative source of information on the world's plant biodiversity

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression