Three-dimensional flagella structures from animals’ closest unicellular relatives, the Choanoflagellates
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Evaluation Summary:
Cryo-electron tomography has been successful at uncovering the details of the eukaryotic cilium/flagellum but there remains a limited number of taxa represented in the studies to date. Pinskey et al. fill this gap with a flagellar structure from choanoflagellates: single-celled organisms that are the closest living relatives of animals. The findings yield many new insights of broad interest to the field, such as the similarity of outer dynein arms and radial spokes to metazoan cilia, the observation of a flagellar vanes, and the presence of mysterious barb structures.
(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. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of flagellar structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the Opisthokonta clade, leaving a sizeable gap in our understanding of flagella evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans’ closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on flagella from the choanoflagellate species Salpingoeca rosetta . We show that the axonemal dyneins, radial spokes, and central pair complex in S. rosetta more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of S. rosetta flagella, including microtubule holes, microtubule inner proteins, and the flagellar vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the flagellar membrane. Together, our findings provide new insights into choanoflagellate biology and flagella evolution between unicellular and multicellular opisthokonts.
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Author Response
Reviewer #1 (Public Review):
Weaknesses:
The data presented here is, on the whole, descriptive. Whilst the descriptive elements are strong and important, more analysis and quantification is required to support the conclusions made in the paper. For example, in contrast to their analysis of the rail-MIP, their assertion that the ciliary vane orientation is linked to the CPC orientation is not backed up by quantification. In addition, this paper does not extensively discuss proteins within the MIP densities and central pair complex in detail, to the extent they can be discussed using the recent structures from Chlamydomonas.
We thank the reviewer for pointing out these areas for improvement, which are addressed. We are grateful for their helpful suggestions, which we have incorporated to the best of our ability to …
Author Response
Reviewer #1 (Public Review):
Weaknesses:
The data presented here is, on the whole, descriptive. Whilst the descriptive elements are strong and important, more analysis and quantification is required to support the conclusions made in the paper. For example, in contrast to their analysis of the rail-MIP, their assertion that the ciliary vane orientation is linked to the CPC orientation is not backed up by quantification. In addition, this paper does not extensively discuss proteins within the MIP densities and central pair complex in detail, to the extent they can be discussed using the recent structures from Chlamydomonas.
We thank the reviewer for pointing out these areas for improvement, which are addressed. We are grateful for their helpful suggestions, which we have incorporated to the best of our ability to improve the quality of the manuscript.
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Evaluation Summary:
Cryo-electron tomography has been successful at uncovering the details of the eukaryotic cilium/flagellum but there remains a limited number of taxa represented in the studies to date. Pinskey et al. fill this gap with a flagellar structure from choanoflagellates: single-celled organisms that are the closest living relatives of animals. The findings yield many new insights of broad interest to the field, such as the similarity of outer dynein arms and radial spokes to metazoan cilia, the observation of a flagellar vanes, and the presence of mysterious barb structures.
(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. Reviewer #2 and Reviewer #3 agreed to share their names with the …
Evaluation Summary:
Cryo-electron tomography has been successful at uncovering the details of the eukaryotic cilium/flagellum but there remains a limited number of taxa represented in the studies to date. Pinskey et al. fill this gap with a flagellar structure from choanoflagellates: single-celled organisms that are the closest living relatives of animals. The findings yield many new insights of broad interest to the field, such as the similarity of outer dynein arms and radial spokes to metazoan cilia, the observation of a flagellar vanes, and the presence of mysterious barb structures.
(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. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Pinskey et al. examine the cilia from the closest unicellular relatives to metazoans, the choanoflagellates. Previous cilia structures come from more distantly-related organisms outside of the opisthokont clade or multicellular metazoans. As such it is not clear whether features that distinguish metazoan cilia, such as central pair shape or dynein motor number in the outer arm dyneins, are a natural consequence of the shift to multicellularity. The authors reveal the ultrastructure of Salpingoeca rosetta, using a combination of cryo-FIB milling and cryo-ET to great effect. Their doublet microtubule and central pair complex structures show that these unicellular organisms contain many of the hallmarks of metazoan cilia, in terms of attached protein densities. The authors identify unique MIPs and features, …
Reviewer #1 (Public Review):
Pinskey et al. examine the cilia from the closest unicellular relatives to metazoans, the choanoflagellates. Previous cilia structures come from more distantly-related organisms outside of the opisthokont clade or multicellular metazoans. As such it is not clear whether features that distinguish metazoan cilia, such as central pair shape or dynein motor number in the outer arm dyneins, are a natural consequence of the shift to multicellularity. The authors reveal the ultrastructure of Salpingoeca rosetta, using a combination of cryo-FIB milling and cryo-ET to great effect. Their doublet microtubule and central pair complex structures show that these unicellular organisms contain many of the hallmarks of metazoan cilia, in terms of attached protein densities. The authors identify unique MIPs and features, such as holes in the microtubule lattice. In addition they describe other unique features of these cilia, notably the striking ciliary vane and barb structures. Their data support their conclusions. The findings presented will interest multiple fields, namely the cilia field, evolutionary biologists working on the origin of multicellularity, and structural biology.
Strengths:
The authors use a series of excellent reconstructions to show convincingly that cilia from the choanoflagellate S. rosetta have many of the hallmarks of metazoan cilia. This is followed by a good description and analysis of the shape and distribution of the rail-MIP and microtubule lattice hole, which are both unique in this structure. In their discussion they hypothesise that the reduction of structural ciliary components seen in multicellular metazoans, compared with cilia of other unicellular organisms, reflects a transition from a requirement for a strong ciliary beat in a harsh open environment to beating in a more protected environment with fewer shear forces. They demonstrate that S. rosetta sit in the middle in terms of structural reduction, supporting this hypothesis. They also provide an excellent description of both the ciliary vane and barb structures found on S. rosetta.
Weaknesses:
The data presented here is, on the whole, descriptive. Whilst the descriptive elements are strong and important, more analysis and quantification is required to support the conclusions made in the paper. For example, in contrast to their analysis of the rail-MIP, their assertion that the ciliary vane orientation is linked to the CPC orientation is not backed up by quantification. In addition, this paper does not extensively discuss proteins within the MIP densities and central pair complex in detail, to the extent they can be discussed using the recent structures from Chlamydomonas.
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Reviewer #2 (Public Review):
This is a lovely study that documents the structure of the flagellum of the choanoflagellate S. rosetta using cryo-electron tomography. The authors have generated an extensive dataset of 54 cryo-electron tomograms, collectively covering >7500 axonemal repeats that were used to generate an average structure. Doing so, they come at a number of novel insights, such as an interestingly animal-like structure for outer dynein arms and radial spokes, the presence of a flagellar vane (known in other choanoflagellates but seen for the first time in S. rosetta), and the discovery of two entirely new structures (barb structures and a "rail-MIP" (Microtubule Inner Protein)). This will be of broad interest to people interested in cilia/flagella and in eukaryotic/metazoan evolution.
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Reviewer #3 (Public Review):
The ultrastructural details of eukaryotic cilium/flagellum architecture have been described in unprecedented resolution in a number of recent cryo-electron microscopy studies. These studies have however focused on a limited number of taxa, and there remain significant gaps in our knowledge to date. In particular, while there have been studies on the cilia of animals (which belong to the Opisthokonta clade) and those of unicellular eukaryotes such as algae and trypanosomes, there has to date been no examination of the cilia of unicellular opisthokonts. Pinskey et al. address this gap by examining the ciliary ultrastructure of choanoflagellates, single-celled organisms that are closely related to sponges.
In this paper, Pinskey et al. document the arrangement of structures in the 96-nm axonemal repeat, noting …
Reviewer #3 (Public Review):
The ultrastructural details of eukaryotic cilium/flagellum architecture have been described in unprecedented resolution in a number of recent cryo-electron microscopy studies. These studies have however focused on a limited number of taxa, and there remain significant gaps in our knowledge to date. In particular, while there have been studies on the cilia of animals (which belong to the Opisthokonta clade) and those of unicellular eukaryotes such as algae and trypanosomes, there has to date been no examination of the cilia of unicellular opisthokonts. Pinskey et al. address this gap by examining the ciliary ultrastructure of choanoflagellates, single-celled organisms that are closely related to sponges.
In this paper, Pinskey et al. document the arrangement of structures in the 96-nm axonemal repeat, noting the similarities and differences with available structures from other taxa. The outer dynein arms, inner dynein arms, radial spokes, microtubule inner proteins and central pair complex are all described closely.
They additionally present a preliminary architecture of the ciliary vane, a brushlike arrangement of filaments outside the cilium that is thought to be involved in generating fluid flows to facilitate feeding. Pinskey et al. also discover the existence of "barbs", four-legged structures studded on the surface of the ciliary membrane, and which may be involved in the generation or maintenance of the ciliary vane.
The methods employed are cutting-edge, and the quality of the data is high, as is its presentation. The work undoubtedly fills a taxonomic gap in our knowledge of the ultrastructure of eukaryotic cilia, and given the choanoflagellates position on the boundary between unicellular/colonial and multicellular life, this will be a very valuable reference point for future studies.
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