Regulation of minimal spindle midzone organization by mitotic kinases.

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis.

Artificial intelligence-supported applications in head and neck cancer radiotherapy treatment planning and dose optimisation.

INTRODUCTION: The aim of this review is to describe how various AI-supported applications are used in head and neck cancer radiotherapy treatment planning, and the impact on dose management in regards to target volume and nearby organs at risk (OARs). METHODS: Literature searches were conducted in databases and publisher portals Pubmed, Science Direct, CINAHL, Ovid, and ProQuest to peer reviewed studies published between 2015 and 2021. RESULTS: Out of 464 potential ones, ten articles covering the topic were selected. The benefit of using deep learning-based methods to automatically segment OARs is that it makes the process more efficient producing clinically acceptable OAR doses. In some cases automated treatment planning systems can outperform traditional systems in dose prediction. CONCLUSIONS: Based on the selected articles, in general AI-based systems produced time savings. Also, AI-based solutions perform at the same level or better than traditional planning systems considering auto-segmentation, treatment planning and dose prediction. However, their clinical implementation into routine standard of care should be carefully validated IMPLICATIONS TO PRACTICE: AI has a primary benefit in reducing treatment planning time and improving plan quality allowing dose reduction to the OARs thereby enhancing patients' quality of life. It has a secondary benefit of reducing radiation therapists' time spent annotating thereby saving their time for e.g. patient encounters.

Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells.

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent "bundling, sliding, and compaction" model of antiparallel midzone bundle formation in the central spindle during late mitosis.

Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

CLIP-170 is essential for MTOC repositioning during T cell activation by regulating dynein localisation on the cell surface.

The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging. Inhibition of CLIP-170 phosphorylation impaired both MTOC repositioning and interleukin-2 (IL-2) expression. T cell stimulation induced some fraction of dynein to colocalise with CLIP-170 and undergo plus-end tracking. Concurrently, it increased dynein in minus-end-directed movement. It also increased dynein relocation to the centre of the contact surface. Dynein not colocalised with CLIP-170 showed both an immobile state and minus-end-directed movement at a velocity in good agreement with the velocity of MTOC repositioning, which suggests that dynein at the immunological synapse may pull the microtubules and the MTOC. Although CLIP-170 is phosphorylated by AMP-activated protein kinase (AMPK) irrespective of stimulation, phosphorylated CLIP-170 is essential for dynein recruitment to plus-end tracking and for dynein relocation. This indicates that dynein relocation results from coexistence of plus-end- and minus-end-directed translocation. In conclusion, CLIP-170 plays an indispensable role in MTOC repositioning and full activation of T cells by regulating dynein localisation.