The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

Rogala, Kacper B.; Dynes, Nicola J.; Hatzopoulos, Georgios N.; Yan, Jun; Pong, Sheng Kai; Robinson, Carol V.; Deane, Charlotte M.; Gönczy, Pierre; Vakonakis, Ioannis

doi:10.7554/elife.07410

Cited by 37 publications

(53 citation statements)

References 78 publications

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“…A cheY gene containing 5′ insertion mutation for cysteine before the wild-type sequence, ( cys ) cheY , was cloned into the enterobacterial expression vector pFloat [ 23 , 24 ], derived from pET30a (Novagen) and engineered to include in frame of its 5′ region a 6xHis (His6) sequence, a small ubiquitin-like modifier (SUMO) solubility tag and a human rhinovirus 3C protease cleavage sequence (LEVLFQ/GP). Formation of correct construct was checked by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule imaging of electroporated dye-labelled CheY in liveEscherichia coli

Paolo

Afanzar

Armitage

et al. 2016

Phil. Trans. R. Soc. B

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For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.This article is part of the themed issue ‘The new bacteriology’.

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Section: Methodsmentioning

confidence: 99%

Single-molecule imaging of electroporated dye-labelled CheY in liveEscherichia coli

Paolo

Afanzar

Armitage

et al. 2016

Phil. Trans. R. Soc. B

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“…A synthetic, codon-optimized DNA fragment (Integrated DNA Technologies) encoding PfHsp70-x residues 424-650 (PfHsp70-x SDB; UniProt K7NTP5) was cloned in the pFLOAT vector (Rogala et al, 2015), which provides an N-terminal His 6 tag separated from the target protein by a Human rhinovirus (HRV) 3C protease cleavage site ( Table 1).…”

Section: Macromolecule Productionmentioning

confidence: 99%

Structure of the substrate-binding domain of Plasmodium falciparum heat-shock protein 70-x

Schmidt

Vakonakis

2020

Acta Crystallogr F Struct Biol Commun

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The malaria parasite Plasmodium falciparum extensively modifies erythrocytes that it invades by exporting a large complement of proteins to the host cell. Among these exported components is a single heat-shock 70 kDa class protein, PfHsp70-x, that supports the virulence and growth rate of the parasite during febrile episodes. The ATP-binding domain of PfHsp70-x has previously been resolved and showed the presence of potentially druggable epitopes that differ from those on human Hsp70 chaperones. Here, the crystallographic structure of the substrate-binding domain (SBD) of PfHsp70-x is presented in complex with a hydrophobic peptide. The PfHsp70-x SBD is shown to be highly similar to the counterpart from a human erythrocytic Hsp70 chaperone. The binding of substrate at the interface between β-sandwich and α-helical subdomains of this chaperone segment is also conserved between the malaria parasite and humans. It is hypothesized that the parasite may partly exploit human chaperones for intra-erythrocytic trafficking and maintenance of its exported proteome.

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“…The critical function of the kinase activity of ZYG-1/Plk4 in promoting SAS-6 oligomerization is not known, although the phosphorylated target is unlikely to be SAS-6 Lettman et al 2013). SAS-5 forms homo-oligomers through two distinct domains, and mutations that disrupt oligomerization prevent centriole duplication (Qiao et al 2012;Rogala et al 2015), although the function of SAS-5 oligomerization in centriole assembly remains unclear.…”

Section: Centriole Assembly: a Higher Resolution Viewmentioning

confidence: 99%

Mitotic Cell Division in Caenorhabditis elegans

Pintard

Bowerman

2019

Genetics

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Mitotic cell divisions increase cell number while faithfully distributing the replicated genome at each division. The Caenorhabditis elegans embryo is a powerful model for eukaryotic cell division. Nearly all of the genes that regulate cell division in C. elegans are conserved across metazoan species, including humans. The C. elegans pathways tend to be streamlined, facilitating dissection of the more redundant human pathways. Here, we summarize the virtues of C. elegans as a model system and review our current understanding of centriole duplication, the acquisition of pericentriolar material by centrioles to form centrosomes, the assembly of kinetochores and the mitotic spindle, chromosome segregation, and cytokinesis. Abstract 35 Centriole Duplication and Centrosome Maturation: Ensuring Fidelity in Bipolar Mitotic Spindle Assembly 37 Centriole disengagement and the initiation of centriole duplication 39 The centriole assembly pathway 39 Centriole assembly: a higher resolution view 42 Limiting centriole duplication by controlling the levels of centriole components 43 PCM assembly dynamics and structure 43 In vitro reconstitution of PCM assembly 45 Kinetochore Assembly, Function, and Regulation 45 Molecular architecture of the C. elegans kinetochore 47 Inner kinetochore proteins: connecting with chromosomal DNA 47 Outer kinetochore proteins: connecting with microtubules 47From lateral to end-on microtubule attachment: cross-talk between RZZ-Spindly and the Ndc80 complex 49 Microtubule attachments and the SAC 49The SAC

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The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

Cited by 37 publications

References 78 publications

Single-molecule imaging of electroporated dye-labelled CheY in liveEscherichia coli

Single-molecule imaging of electroporated dye-labelled CheY in liveEscherichia coli

Structure of the substrate-binding domain of Plasmodium falciparum heat-shock protein 70-x

Mitotic Cell Division in Caenorhabditis elegans

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