Evolutionary conservation of centriole rotational asymmetry in the human centrosome
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Evaluation Summary:
This paper shows that centrioles of the human centrosome are rotationally asymmetric, a feature previously known only from centrioles in flagellated protists and in multi-ciliated cells. The authors, identify LRRCC1, implicated in human ciliopathy, as a component that localizes asymmetrically on one side of the distal lumen, contributing to proper centriole structure, ciliary assembly and ciliary signaling. This work is of interest to cell biologists studying how centriole assembly and function is linked to human disease.
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Abstract
Centrioles are formed by microtubule triplets in a ninefold symmetric arrangement. In flagellated protists and animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the ‘acorn.’ Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly, and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.
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Evaluation Summary:
This paper shows that centrioles of the human centrosome are rotationally asymmetric, a feature previously known only from centrioles in flagellated protists and in multi-ciliated cells. The authors, identify LRRCC1, implicated in human ciliopathy, as a component that localizes asymmetrically on one side of the distal lumen, contributing to proper centriole structure, ciliary assembly and ciliary signaling. This work is of interest to cell biologists studying how centriole assembly and function is linked to human disease.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this work the authors investigate whether centrioles of the human centrosome, which are composed of 9 symmetrically arranged microtubule triplets, may display rotational asymmetry. This feature was previously described for centrioles in flagellated protists and in multi-ciliated cells, whereas human centrioles in the centrosome do not display any obvious rotational asymmetry. However, by beautiful ultra structure expansion microscopy imaging they show asymmetric localization on one side of the distal centriole lumen of the centriole protein LRRCC1, the ortholog of a protein originally shown in flagellate green algae as being asymmetrically localized. They also show that LRRCC1 affects the recruitment of another centriole protein, C2CD3, which also displays asymmetric, albeit not identical, distribution in …
Reviewer #1 (Public Review):
In this work the authors investigate whether centrioles of the human centrosome, which are composed of 9 symmetrically arranged microtubule triplets, may display rotational asymmetry. This feature was previously described for centrioles in flagellated protists and in multi-ciliated cells, whereas human centrioles in the centrosome do not display any obvious rotational asymmetry. However, by beautiful ultra structure expansion microscopy imaging they show asymmetric localization on one side of the distal centriole lumen of the centriole protein LRRCC1, the ortholog of a protein originally shown in flagellate green algae as being asymmetrically localized. They also show that LRRCC1 affects the recruitment of another centriole protein, C2CD3, which also displays asymmetric, albeit not identical, distribution in the distal centriole lumen. In addition, using partial depletion, the authors provide data implicating LRRCC1 in proper centriole architecture, primary cilium assembly and ciliary signaling.
The most important contribution of the paper is the demonstration that human centrioles are generally asymmetric, contrary to what is commonly believed, and suggesting that this is an evolutionary conserved feature. The fact that the identified asymmetrically localized protein LRRCC1 is implicated in human ciliopathy suggests that the asymmetry is functionally important. Unfortunately, in functional studies the authors achieved only partial LRRCC1 depletion and could not achieve rescue, presumably due to toxicity associated with altered LRRCC1 levels. While the observed phenotypes were relatively mild, the data using CRISPR/Cas9 genome editing as well as RNAi still support the notion that asymmetry is functionally important.
Together this is a very well presented study, executed with high technical quality, that introduces rotational asymmetry as an important structural and functional feature of centrioles of the human centrosome. -
Reviewer #2 (Public Review):
The observation that human somatic centrioles are not molecularly rotationally symmetric, despite their structural symmetry, and that this asymmetry mediates appendage formation and ciliation is intriguing. The authors provide a fair evidence that LRRCC1, human ortholog of Vfl1, which is responsible for imparting rotational centriole asymmetry in flagellated organisms, localizes asymmetrically within mouse ependymal ciliated cells, and human RPE1. In mouse cells, LRRCC1 localizes opposite to the basal foot in ciliated centrioles. However, inconsistencies in the pattern and the levels of LRRCC1 in RPE1 across figure panels need to be clarified. The authors further demonstrate that discovered rotational asymmetry is not linked to a role in centriole duplication and there are no concerns regarding this finding.
Reviewer #2 (Public Review):
The observation that human somatic centrioles are not molecularly rotationally symmetric, despite their structural symmetry, and that this asymmetry mediates appendage formation and ciliation is intriguing. The authors provide a fair evidence that LRRCC1, human ortholog of Vfl1, which is responsible for imparting rotational centriole asymmetry in flagellated organisms, localizes asymmetrically within mouse ependymal ciliated cells, and human RPE1. In mouse cells, LRRCC1 localizes opposite to the basal foot in ciliated centrioles. However, inconsistencies in the pattern and the levels of LRRCC1 in RPE1 across figure panels need to be clarified. The authors further demonstrate that discovered rotational asymmetry is not linked to a role in centriole duplication and there are no concerns regarding this finding.
To unravel the functional role of LRRCC1 in human cells, the authors generate three CRISPR clones with decreased levels of LRRCC1. These clones have decreased rate of ciliation and perturbed ciliary signaling. As a possible cause for ciliary abnormalities, the authors suggest that LRRCC1, together with distal protein C2CD3, regulates the assembly of centriole distal ends and distal appendages. However, LRRCC1 is only partially removed, and only one of three clones forms slightly longer centrioles, despite having similar levels of LRRCC1 to other clones. A very limited analysis of centriole ultrastructure in this clone has been provided, showing one centriole pair with one longer than average and possibly structurally aberrant centriole. So, it remains unclear whether this information is relevant to the observed issues with ciliation and how lack of LRRCC1 affects centriole distal end in general. The authors also suggest that localization of distal appendage protein Cep164 is perturbed after LRRCC1 downregulation. This analysis would need to be extended to other appendage proteins.
Finally, the authors propose that LRRCC1 affects localization of another distal centriole protein C2CD3. They further suggest that C2CD3 is also asymmetrically localized within centriole distal lumen, where it partially colocalizes with LRRCC1. However, the claims regarding C2CD3 asymmetry and its disorganization in 1.1 and 1.9 clones would need further evidence. -
Reviewer #3 (Public Review):
The authors make the important discovery that LRRCC1 localization breaks the radial symmetry of the animal centriole, preferentially associating with two consecutive triplets opposite the basal foot. LRRCC1 partially co-localizes with another centriolar protein C2CD3. Depletion of LRRCC1 altered distal appendage production and ciliary recruitment of Smoothened, but whether these requirements reflect the asymmetric nature of its centriole localization remains unclear.
One of the conclusions conflates two observations: the authors state that they "uncover the unanticipated rotational asymmetry of centrioles in the human centrosome and show that this property is connected to the assembly and function of primary cilia." The rotational asymmetry is an advance, but whether it is this asymmetry that is important to …
Reviewer #3 (Public Review):
The authors make the important discovery that LRRCC1 localization breaks the radial symmetry of the animal centriole, preferentially associating with two consecutive triplets opposite the basal foot. LRRCC1 partially co-localizes with another centriolar protein C2CD3. Depletion of LRRCC1 altered distal appendage production and ciliary recruitment of Smoothened, but whether these requirements reflect the asymmetric nature of its centriole localization remains unclear.
One of the conclusions conflates two observations: the authors state that they "uncover the unanticipated rotational asymmetry of centrioles in the human centrosome and show that this property is connected to the assembly and function of primary cilia." The rotational asymmetry is an advance, but whether it is this asymmetry that is important to assembly and function of cilia or some other aspect of LRRCC1 function remains unclear. Said another way, while LRRCC1 and C2CD3 are localized asymmetrically at centrioles, there is no test of whether their asymmetry directly contributes to the assembly and function of primary cilia.
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