Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains microtubule-bound states by slowing ATP-binding resulting in high-force production at both homotetramer ends.
Two atomic coordinate files for Dm-KLP61F motor ATP-like MT(alpha-beta-tubulin) model is available at Protein Data Bank (PDB-ID: #XXXX. The Dm-KLP61F motor nucleotide-free MT(alpha-beta-tubulin) asymmetric unit Protein Data BankPDB-ID: #XXXXThe refined Dm-KLP61F motor AMPPNP MT cryo-EM map is available at the Electron microscopy Data bank (EMBD) EMDB ID:#XXXXand the Dm-KLP61F motor-tail nucleotide free MT cryo-EM map is available at Electron microscopy Data bank (EMBD) EMDB-iD:#XXXXDm-KLP61F motor-tail nucleotide free MT cryo-EM map (focused 3D-classification map) is available at the Electron microscopy Data bank (EMBD)EMDB-ID:#XXXX
- Jawdat Al-Bassam
- Jawdat Al-Bassam
- Jason Stumpff
- Jason Stumpff
- Larisa Gheber
- Larisa Gheber
- Richard J McKenney
- Ron Milligan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom
© 2020, Bodrug et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Having its genome makes the mitochondrion a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule of about 16 kb coding for 37 genes. Mutations, including deletions in the mitochondrial genome, can culminate in different human diseases. Mapping the deletion junctions suggests that the breakpoints are generally seen at hotspots. ‘9 bp deletion’ (8271–8281), seen in the intergenic region of cytochrome c oxidase II/tRNALys, is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with ‘9 bp deletion’ when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Therefore, we uncover a new role for Endonuclease G in generating mtDNA deletions, which depends on the formation of G4 DNA within the mitochondrial genome. In summary, we identify a novel property of Endonuclease G, besides its role in apoptosis and the recently described ‘elimination of paternal mitochondria during fertilisation.
Obesity is generally associated with insulin resistance in liver and muscle and increased risk of developing type 2 diabetes, however there is a population of obese people that remain insulin sensitive. Similarly, recent work suggests that mice fed high carbohydrate diets can become obese without apparent glucose intolerance. To investigate this phenomenon further, we fed mice either a high fat (Hi-F) or high starch (Hi-ST) diet and measured adiposity, glucose tolerance, insulin sensitivity, and tissue lipids compared to control mice fed a standard laboratory chow. Both Hi-ST and Hi-F mice accumulated a similar amount of fat and tissue triglyceride compared to chow-fed mice. However, while Hi-F diet mice developed glucose intolerance as well as liver and muscle insulin resistance (assessed via euglycaemic/hyperinsulinaemic clamp), obese Hi-ST mice maintained glucose tolerance and insulin action similar to lean, chow-fed controls. This preservation of insulin action despite obesity in Hi-ST mice was associated with differences in de novo lipogenesis and levels of C22:0 ceramide in liver and C18:0 ceramide in muscle. This indicates that dietary manipulation can influence insulin action independently of the level of adiposity and that the presence of specific ceramide species correlates with these differences.