A Structural Study of Conserved Centriole Duplication Machinery

Centrioles are microtubule-based cylindrical structures that act within organelles responsible for nucleating polarized microtubule networks. Centrioles have an inherent nine-fold radial symmetry with species-dependent dimensions. Despite the fact that centrioles have been described by biologists for over a century, the process by which cells license and assemble nascent centrioles has only recently started to come to light. It is now known that centrioles, like DNA, duplicate via a cell cycle-regulated mechanism, and that a core set of five conserved proteins is necessary for centriole duplication. Polo-like Kinase 4 (Plk4) is a highly conserved serine/threonine kinase required for centriole duplication licensing. Plk4, along with all other Plk family members, contains arrayed Polo Box (PB) domains, a motif that undergoes hetero- or homo-dimerization to bind targets, localize to subcellular structures, and regulate kinase activity; however, the mechanism by which Plk4 employs its PBs to license centriole duplication has been unclear. Here, we harness x-ray crystallography, biochemistry, and cell biology to show that Drosophila melanogaster Plk4 contains three complete PB domains with distinct functions. The first two, PB1-PB2, form a homodimer in trans in both crystallographic form and in solution. We use in vitro pulldowns to demonstrate that PB1-PB2 are collectively needed to bind Asterless, a centriole scaffolding protein that localizes FL Plk4 to centrioles. Additionally, the PB1-PB2 homodimer is required for downregulation of the FL molecule, limiting centriole duplication. Finally, we use D.m. S2 cells to show that PB1-PB2 localizes to centrioles via conserved electrostatic interactions. The C-terminal PB domain, PB3, also forms a canonical PB fold, yet it shows species-dependent architecture and oligomerization states, demonstrating that PB3 is a structurally variable domain with species-dependent functions. Further work examines the role of a dynein light chain (LC8) in oligomerizing Anastral-2 (Ana2), a downstream centriole component that is a candidate for Plk4 phosphorylation. LC8-based Ana2 tetramerization has further implications for the role of Ana2 during centriole duplication. Collectively, our work delineates novel domains and interactions in the fundamental centriole licensing and assembly proteins Plk4 and Ana2, and implicates conserved mechanisms in centriole biogenesis.Doctor of Philosoph

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156–251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ∼34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis

The Structure of the Plk4 Cryptic Polo Box Reveals Two Tandem Polo Boxes Required for Centriole Duplication

Centrioles are key microtubule polarity determinants. Centriole duplication is tightly controlled to prevent cells from developing multipolar spindles, a situation that promotes chromosomal instability. A conserved component in the duplication pathway is Plk4, a polo kinase family member that localizes to centrioles in M/G1. To limit centriole duplication, Plk4 levels are controlled through trans-autophosphorylation that primes ubiquitination. In contrast to Plks 1–3, Plk4 possesses a unique central region called the “cryptic polo box”. Here, we present the crystal structure of this region at 2.3 Å resolution. Surprisingly, the structure reveals two tandem, homodimerized polo boxes, PB1-PB2, that form a unique, winged architecture. The full PB1-PB2 cassette is required for binding the centriolar protein Asterless as well as robust centriole targeting. Thus, with its C-terminal polo box (PB3), Plk4 has a triple polo box architecture that facilitates oligomerization, targeting, and promotes trans-autophosphorylation, limiting centriole duplication to once per cell cycle

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease