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Multiciliated Cells: Rise and Fall of the Deuterosomes
TICB 1582 No. of Pages 3
Trends in Cell Biology
Spotlight
Multiciliated Cells: Rise and Fall of the Deuterosomes Dheeraj Rayamajhi1,2 and Sudipto Roy1,2,3,* Esoteric organelles called deuterosomes have been implicated in the explosive production of hundreds of basal bodies in multiciliated cells (MCCs). A new study by Meunier, Holland, and colleagues now shows that deuterosomes are dispensable, re-igniting the quest for mechanisms driving basal body biogenesis in this specialized ciliated cell type. MCCs, with their apical arrays of motile cilia, drive fluid movement over epithelia [1]. For example, MCCs within the airways clear mucus and those lining the ependyma of the brain ventricles facilitate cerebrospinal fluid flow. Formation of multiple motile cilia requires a large number of centrioles to be first generated in postmitotic MCC precursors, which later convert into ciliary basal bodies (Figure 1A). Studies over the years have postulated two pathways for centriole amplification in MCCs: the centrioledependent (CD) and deuterosomedependent (DD) pathways. As the name suggests, the first pathway involves centrosomal (parental) centrioles for templating procentriole biogenesis. The second pathway is thought to rely on abstruse electron-dense fibrogranular ring-like structures termed deuterosomes. Discovered through electron microscopic studies in the late 1960s and early 1970s [2], deuterosomes are unique to MCCs, and arise during the early centriole amplification stage, to disappear once amplification is completed. The DD pathway is considered to be the major contributor of centriole production in MCCs; about 90%
of centrioles in mammalian MCCs are cur- that Deup1 is a deuterosome-specific rently believed to arise via the DD pathway. structural protein; the first such molecular component of the deuterosome to be Despite the purported importance of the characterized, and using knockdown deuterosomes, there has been an ongoing strategies in cultured mouse tracheal controversy regarding their origins. Some MCCs (mTECS) and Xenopus embryos researchers argue that the organelles are (which differentiate MCCs on their skin), derived from parental centrioles, while they found it to be a critical determinant others consider a de novo origin. Recent of deuterosome formation [7]. To further findings of Zhao et al. from the Zhu labora- investigate the DD pathway, Mercey et al. tory support the second theory [3]. instead utilized a stable genetic mutation Inhibition of Plk4 (a kinase essential for representing a null allele of Deup1. parental centriole duplication in cycling Confirming the observation of Zhao et al. cells) in cultured mouse ependymal cells [7], Mercey and colleagues found that dif(mEPCs), which rendered them devoid ferentiating MCCs in Deup1 mutant mice, of the parental centrioles, did not derail analyzed in vivo as well as ex vivo/in vitro, deuterosome formation or multiple basal were devoid of deuterosomes. Nonethebody production (Figure 1B). The authors less, and unlike what was reported by also demonstrated that under such cir- Zhao et al. [7], the mutant cells were able cumstances, deuterosomes were the nu- to produce the full complement of basal cleation centers for nascent procentrioles. bodies and differentiate into functional Similar observations were made in two MCCs. Extension of this investigation to other independent studies [4,5], providing morpholino-mediated Deup1 knockedstrong evidence in support of the view down Xenopus embryos also revealed no that MCCs can produce basal bodies with- effect on centriole production. out parental centrioles, via the DD pathway. With centrioles being dispensable, the The authors next examined the dynamics role of the DD pathway in MCC basal of procentriole formation in Deup1-/body biogenesis assumed an even greater MCC precursors. Three stages have significance. been attributed to this process in the wild type : the amplification stage in However, the latest report, that of Mercey which procentrioles form on centriolar et al. published in Nature Cell Biology, and deuterosomal platforms; the growth delivers a fatal blow to the deuterosomes stage in which procentrioles mature; [6]. This work, a collaborative effort from and the disengagement stage in which the Meunier and Holland laboratories, the mature centrioles separate, migrate shows that MCCs can generate the and dock at the apical plasma membrane correct numbers of basal bodies without [8]. Live imaging of ependymal cells from deuterosomes, and more surprisingly, Deup-/− and control mice, which also even in the combined absence of expressed a GFP-tagged version of the deuterosomes and parental centrioles, centriolar protein Centrin-2, revealed that and tentatively implicate the pericentriolar Deup1 mutants preserved the essentials cloud as the source of procentriole of stepwise centriolar amplification dyassembly. namics. The only variations observed were largely restricted to the spatial Earlier pioneering work from the Zhu labo- arrangement of procentrioles, which ratory identified Deup1 (deuterosomal appeared to bud off all along the length protein 1), as a paralog of the centriolar of the parental centrioles, as well as an protein Cep63, that is expressed specifi- increase in procentriole numbers in the cally in differentiating MCCs. They showed vicinity of the parental centrioles. This Trends in Cell Biology, Month 2020, Vol. xx, No. xx
1
Trends in Cell Biology
Trends in Cell Biology
Figure 1. Multiciliated Cells (MCCs) and Different Pathways for Basal Body Generation. (A) MCCs derived from mouse tracheal epithelial cells (mTECs) in culture. Cilia were labeled with antiacetylated tubulin (green) and basal bodies with anti-γ-tubulin antibodies (red), respectively. Nuclei were highlighted with DAPI (blue). Scale bar = 5 μm. (B) Centriole-dependent (CD) pathway. (C) Deuterosome-dependent (DD) pathway. (D) De novo pathway. Abbreviations: PCM, pericentriolar material.
increase, as the authors suggest, is likely decrease in centriole numbers, respecdue to an upregulation of the activity of tively, a finding that was confirmed by the combined knock-down of Cep63 and the CD pathway. Deup1 in Xenopus epidermal MCCs. The Given that Deup1 is a paralog of Cep63, results again contradict the findings of any compensation from Cep63 was Zhao et al. [7], where a 90% decrease next evaluated. However, mTECs and in centriole numbers was recorded in mEPCs from Deup1-/-; Cep63-/- double mTECs knocked down for Deup1 and mutants showed from none to only a slight Cep63. Electron and video microscopy 2
Trends in Cell Biology, Month 2020, Vol. xx, No. xx
revealed absence of any major structural or functional abnormalities in the basal bodies and their cilia. Assuming that parental centrioles likely compensated for deuterosome loss in Deup1-/- MCCs, in a final experiment, the authors investigated the consequences of treating Deup1-/mEPCs with a Plk4 inhibitor, used in the earlier studies to generate parental
Trends in Cell Biology
centriole-less MCCs [3–5], so that both the DD as well as the CD pathways were inactivated. Strikingly, there was no significant difference in centriole numbers in Deup1-/- cells even in the absence of one or both of the centrioles. From where do all of the centrioles arise in such CD- and DD-pathway-inactivated MCCs? Small clusters of procentrioles were observed in the vicinity of the pericentriolar material (PCM) and foci of fibrogranular material concentrated near the nuclear membrane, leading the authors to tentatively conclude that this could be the minimal microenvironment sufficient for centriole biogenesis. In line with this hypothesis, several previous studies have demonstrated the de novo generation of centrioles in centriole-less non-MCC cell types, perhaps from the PCM cloud [9]. Furthermore, Ito et al. have recently shown that the PCM component Pericentrin can indeed recruit core centriolar proteins, such as Sas-6, and promote centriole biogenesis [10]. While further investigation into the composition of the PCM/fibrogranular complex will undoubtedly be required to reveal the precise mechanisms that drive basal body production in MCCs devoid of parental centrioles and deuterosomes, nevertheless, the elegant analysis of Mercey et al. [6] puts to rest the ongoing debate on the necessity of parental centrioles and the deuterosomes and their relative contributions to basal body biogenesis. Going forward, we need to contend between two possibilities. First, during normal MCC differentiation, centrioles
arise de novo from the PCM, and parental Acknowledgments centrioles and deuterosomes merely func- We thank H. Lu for providing images for Figure 1A. tion as secondary (nonessential) sites of D.R. is supported by a Singapore International Graduate Award (SINGA) studentship. Work on centriprocentriole nucleation, the latter also oles and cilia in the S.R. laboratory is funded by ferrying newly formed procentrioles away the Agency for Science, Technology, and Research from the PCM. Better microscopy, that (A*STAR) of Singapore. can capture the initiating events of centriole biogenesis, could vindicate this view. Second, parental centrioles and 1 Institute of Molecular and Cell Biology, Proteos, 61 Biopolis deuterosomes are indeed the sources of Drive, Singapore 138673 2 centriole production in wild-type MCCs, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 but without them, a compensatory (likely 3Department of Pediatrics, Yong Loo Ling School of Medicine, de novo) pathway is activated in the National University of Singapore, 1E Kent Ridge Road, Singapore 119288 PCM. Compensatory pathways have been increasingly shown to get activated *Correspondence: in situations of chronic loss of gene activ- [email protected] (S. Roy). ity. However, arguing against this possibil- https://doi.org/10.1016/j.tcb.2020.02.003 ity is the observation made in the Mercey © 2020 Elsevier Ltd. All rights reserved. et al. study [6]: that acute loss of Deup1 using RNAi as well as morpholinos did References 1. Brooks, E.R. and Wallingford, J.B. (2014) Multiciliated not affect basal body production in mamcells. Curr. Biol. 24, R973–R982 malian or frog MCCs, respectively; again, 2. Dirksen, E.R. (1971) Centriole morphogenesis in developing ciliated epithelium of the mouse oviduct. J. Cell Biol. contrary to what was reported earlier 51, 286–302 from the Zhu group [7]. The reason for 3. Zhao, H. et al. (2019) Parental centrioles are dispensable for deuterosome formation and function during basal this discrepancy could arise from differbody amplification. EMBO Rep. Published online March 4, 2019. http://dx.doi.org/10.15252/embr.201846735 ences in the strengths of the knockdown 4. Nanjundappa, R. et al. (2019) Regulation of cilia abunapproaches used in the two studies, dance in multiciliated cells. Elife Published online April 26, 2019. https://doi.org/10.7554/eLife.44039 since such strategies for gene inactivation 5. Mercey, O. et al. (2019) Dynamics of centriole amplification are always prone to subjective variations. in centrosome-depleted brain multiciliated progenitors. How MCCs generate hundreds of basal bodies have captivated the interest of cell biologists for decades, and lately, with the molecular characterization of the DD pathway, it seemed that we were making significant headway. Now the demise of the deuterosomes does take us into reverse gear, and we have to again look ahead to the coming years for new insights to unravel the intricacies of this intriguing phenomenon.
Sci. Rep. 9, 13060 Mercey, O. et al. (2019) Massive centriole production can occur in the absence of deuterosomes in multiciliated cells. Nat. Cell Biol. 21, 1544–1552 7. Zhao, H. et al. (2013) The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. Nat. Cell Biol. 15, 1434–1444 8. Spassky, N. and Meunier, A. (2017) The development and functions of multiciliated epithelia. Nat. Rev. Mol. Cell Biol. 18, 423–436 9. Loncarek, J. et al. (2007) Centriole biogenesis: a tale of two pathways. Nat. Cell Biol. 9, 736–738 10. Ito, D. et al. (2019) Pericentrin-mediated SAS-6 recruitment promotes centriole assembly. Elife Published online June 11, 2019. https://doi.org/10.7554/eLife.41418 6.
Trends in Cell Biology, Month 2020, Vol. xx, No. xx
3
Trends in Cell Biology
Spotlight
Multiciliated Cells: Rise and Fall of the Deuterosomes Dheeraj Rayamajhi1,2 and Sudipto Roy1,2,3,* Esoteric organelles called deuterosomes have been implicated in the explosive production of hundreds of basal bodies in multiciliated cells (MCCs). A new study by Meunier, Holland, and colleagues now shows that deuterosomes are dispensable, re-igniting the quest for mechanisms driving basal body biogenesis in this specialized ciliated cell type. MCCs, with their apical arrays of motile cilia, drive fluid movement over epithelia [1]. For example, MCCs within the airways clear mucus and those lining the ependyma of the brain ventricles facilitate cerebrospinal fluid flow. Formation of multiple motile cilia requires a large number of centrioles to be first generated in postmitotic MCC precursors, which later convert into ciliary basal bodies (Figure 1A). Studies over the years have postulated two pathways for centriole amplification in MCCs: the centrioledependent (CD) and deuterosomedependent (DD) pathways. As the name suggests, the first pathway involves centrosomal (parental) centrioles for templating procentriole biogenesis. The second pathway is thought to rely on abstruse electron-dense fibrogranular ring-like structures termed deuterosomes. Discovered through electron microscopic studies in the late 1960s and early 1970s [2], deuterosomes are unique to MCCs, and arise during the early centriole amplification stage, to disappear once amplification is completed. The DD pathway is considered to be the major contributor of centriole production in MCCs; about 90%
of centrioles in mammalian MCCs are cur- that Deup1 is a deuterosome-specific rently believed to arise via the DD pathway. structural protein; the first such molecular component of the deuterosome to be Despite the purported importance of the characterized, and using knockdown deuterosomes, there has been an ongoing strategies in cultured mouse tracheal controversy regarding their origins. Some MCCs (mTECS) and Xenopus embryos researchers argue that the organelles are (which differentiate MCCs on their skin), derived from parental centrioles, while they found it to be a critical determinant others consider a de novo origin. Recent of deuterosome formation [7]. To further findings of Zhao et al. from the Zhu labora- investigate the DD pathway, Mercey et al. tory support the second theory [3]. instead utilized a stable genetic mutation Inhibition of Plk4 (a kinase essential for representing a null allele of Deup1. parental centriole duplication in cycling Confirming the observation of Zhao et al. cells) in cultured mouse ependymal cells [7], Mercey and colleagues found that dif(mEPCs), which rendered them devoid ferentiating MCCs in Deup1 mutant mice, of the parental centrioles, did not derail analyzed in vivo as well as ex vivo/in vitro, deuterosome formation or multiple basal were devoid of deuterosomes. Nonethebody production (Figure 1B). The authors less, and unlike what was reported by also demonstrated that under such cir- Zhao et al. [7], the mutant cells were able cumstances, deuterosomes were the nu- to produce the full complement of basal cleation centers for nascent procentrioles. bodies and differentiate into functional Similar observations were made in two MCCs. Extension of this investigation to other independent studies [4,5], providing morpholino-mediated Deup1 knockedstrong evidence in support of the view down Xenopus embryos also revealed no that MCCs can produce basal bodies with- effect on centriole production. out parental centrioles, via the DD pathway. With centrioles being dispensable, the The authors next examined the dynamics role of the DD pathway in MCC basal of procentriole formation in Deup1-/body biogenesis assumed an even greater MCC precursors. Three stages have significance. been attributed to this process in the wild type : the amplification stage in However, the latest report, that of Mercey which procentrioles form on centriolar et al. published in Nature Cell Biology, and deuterosomal platforms; the growth delivers a fatal blow to the deuterosomes stage in which procentrioles mature; [6]. This work, a collaborative effort from and the disengagement stage in which the Meunier and Holland laboratories, the mature centrioles separate, migrate shows that MCCs can generate the and dock at the apical plasma membrane correct numbers of basal bodies without [8]. Live imaging of ependymal cells from deuterosomes, and more surprisingly, Deup-/− and control mice, which also even in the combined absence of expressed a GFP-tagged version of the deuterosomes and parental centrioles, centriolar protein Centrin-2, revealed that and tentatively implicate the pericentriolar Deup1 mutants preserved the essentials cloud as the source of procentriole of stepwise centriolar amplification dyassembly. namics. The only variations observed were largely restricted to the spatial Earlier pioneering work from the Zhu labo- arrangement of procentrioles, which ratory identified Deup1 (deuterosomal appeared to bud off all along the length protein 1), as a paralog of the centriolar of the parental centrioles, as well as an protein Cep63, that is expressed specifi- increase in procentriole numbers in the cally in differentiating MCCs. They showed vicinity of the parental centrioles. This Trends in Cell Biology, Month 2020, Vol. xx, No. xx
1
Trends in Cell Biology
Trends in Cell Biology
Figure 1. Multiciliated Cells (MCCs) and Different Pathways for Basal Body Generation. (A) MCCs derived from mouse tracheal epithelial cells (mTECs) in culture. Cilia were labeled with antiacetylated tubulin (green) and basal bodies with anti-γ-tubulin antibodies (red), respectively. Nuclei were highlighted with DAPI (blue). Scale bar = 5 μm. (B) Centriole-dependent (CD) pathway. (C) Deuterosome-dependent (DD) pathway. (D) De novo pathway. Abbreviations: PCM, pericentriolar material.
increase, as the authors suggest, is likely decrease in centriole numbers, respecdue to an upregulation of the activity of tively, a finding that was confirmed by the combined knock-down of Cep63 and the CD pathway. Deup1 in Xenopus epidermal MCCs. The Given that Deup1 is a paralog of Cep63, results again contradict the findings of any compensation from Cep63 was Zhao et al. [7], where a 90% decrease next evaluated. However, mTECs and in centriole numbers was recorded in mEPCs from Deup1-/-; Cep63-/- double mTECs knocked down for Deup1 and mutants showed from none to only a slight Cep63. Electron and video microscopy 2
Trends in Cell Biology, Month 2020, Vol. xx, No. xx
revealed absence of any major structural or functional abnormalities in the basal bodies and their cilia. Assuming that parental centrioles likely compensated for deuterosome loss in Deup1-/- MCCs, in a final experiment, the authors investigated the consequences of treating Deup1-/mEPCs with a Plk4 inhibitor, used in the earlier studies to generate parental
Trends in Cell Biology
centriole-less MCCs [3–5], so that both the DD as well as the CD pathways were inactivated. Strikingly, there was no significant difference in centriole numbers in Deup1-/- cells even in the absence of one or both of the centrioles. From where do all of the centrioles arise in such CD- and DD-pathway-inactivated MCCs? Small clusters of procentrioles were observed in the vicinity of the pericentriolar material (PCM) and foci of fibrogranular material concentrated near the nuclear membrane, leading the authors to tentatively conclude that this could be the minimal microenvironment sufficient for centriole biogenesis. In line with this hypothesis, several previous studies have demonstrated the de novo generation of centrioles in centriole-less non-MCC cell types, perhaps from the PCM cloud [9]. Furthermore, Ito et al. have recently shown that the PCM component Pericentrin can indeed recruit core centriolar proteins, such as Sas-6, and promote centriole biogenesis [10]. While further investigation into the composition of the PCM/fibrogranular complex will undoubtedly be required to reveal the precise mechanisms that drive basal body production in MCCs devoid of parental centrioles and deuterosomes, nevertheless, the elegant analysis of Mercey et al. [6] puts to rest the ongoing debate on the necessity of parental centrioles and the deuterosomes and their relative contributions to basal body biogenesis. Going forward, we need to contend between two possibilities. First, during normal MCC differentiation, centrioles
arise de novo from the PCM, and parental Acknowledgments centrioles and deuterosomes merely func- We thank H. Lu for providing images for Figure 1A. tion as secondary (nonessential) sites of D.R. is supported by a Singapore International Graduate Award (SINGA) studentship. Work on centriprocentriole nucleation, the latter also oles and cilia in the S.R. laboratory is funded by ferrying newly formed procentrioles away the Agency for Science, Technology, and Research from the PCM. Better microscopy, that (A*STAR) of Singapore. can capture the initiating events of centriole biogenesis, could vindicate this view. Second, parental centrioles and 1 Institute of Molecular and Cell Biology, Proteos, 61 Biopolis deuterosomes are indeed the sources of Drive, Singapore 138673 2 centriole production in wild-type MCCs, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 but without them, a compensatory (likely 3Department of Pediatrics, Yong Loo Ling School of Medicine, de novo) pathway is activated in the National University of Singapore, 1E Kent Ridge Road, Singapore 119288 PCM. Compensatory pathways have been increasingly shown to get activated *Correspondence: in situations of chronic loss of gene activ- [email protected] (S. Roy). ity. However, arguing against this possibil- https://doi.org/10.1016/j.tcb.2020.02.003 ity is the observation made in the Mercey © 2020 Elsevier Ltd. All rights reserved. et al. study [6]: that acute loss of Deup1 using RNAi as well as morpholinos did References 1. Brooks, E.R. and Wallingford, J.B. (2014) Multiciliated not affect basal body production in mamcells. Curr. Biol. 24, R973–R982 malian or frog MCCs, respectively; again, 2. Dirksen, E.R. (1971) Centriole morphogenesis in developing ciliated epithelium of the mouse oviduct. J. Cell Biol. contrary to what was reported earlier 51, 286–302 from the Zhu group [7]. The reason for 3. Zhao, H. et al. (2019) Parental centrioles are dispensable for deuterosome formation and function during basal this discrepancy could arise from differbody amplification. EMBO Rep. Published online March 4, 2019. http://dx.doi.org/10.15252/embr.201846735 ences in the strengths of the knockdown 4. Nanjundappa, R. et al. (2019) Regulation of cilia abunapproaches used in the two studies, dance in multiciliated cells. Elife Published online April 26, 2019. https://doi.org/10.7554/eLife.44039 since such strategies for gene inactivation 5. Mercey, O. et al. (2019) Dynamics of centriole amplification are always prone to subjective variations. in centrosome-depleted brain multiciliated progenitors. How MCCs generate hundreds of basal bodies have captivated the interest of cell biologists for decades, and lately, with the molecular characterization of the DD pathway, it seemed that we were making significant headway. Now the demise of the deuterosomes does take us into reverse gear, and we have to again look ahead to the coming years for new insights to unravel the intricacies of this intriguing phenomenon.
Sci. Rep. 9, 13060 Mercey, O. et al. (2019) Massive centriole production can occur in the absence of deuterosomes in multiciliated cells. Nat. Cell Biol. 21, 1544–1552 7. Zhao, H. et al. (2013) The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. Nat. Cell Biol. 15, 1434–1444 8. Spassky, N. and Meunier, A. (2017) The development and functions of multiciliated epithelia. Nat. Rev. Mol. Cell Biol. 18, 423–436 9. Loncarek, J. et al. (2007) Centriole biogenesis: a tale of two pathways. Nat. Cell Biol. 9, 736–738 10. Ito, D. et al. (2019) Pericentrin-mediated SAS-6 recruitment promotes centriole assembly. Elife Published online June 11, 2019. https://doi.org/10.7554/eLife.41418 6.
Trends in Cell Biology, Month 2020, Vol. xx, No. xx
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