- Email: [email protected]
Basal Feet: Walking to the Discovery of a Novel Hybrid Cilium
ll
Previews transdifferentiation are back in style. Nat. Rev. Mol. Cell Biol. 17, 413–425. Miao, Z.-F., Lewis, M.A., Cho, C.J., AdkinsThreats, M., Park, D., Brown, J.W., Sun, J.-X., Burclaff, J.R., Kennedy, S., Lu, J., et al. (2020). A dedicated evolutionarily conserved molecular network licenses differentiated cells to return to the cell cycle. Dev. Cell 55, this issue, 178–194. Rodgers, J.T., King, K.Y., Brett, J.O., Cromie, M.J., Charville, G.W., Maguire, K.K., Brunson, C.,
Mastey, N., Liu, L., Tsai, C.-R., et al. (2014). mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510, 393–396. Willet, S.G., Lewis, M.A., Miao, Z., Liu, D., Radyk, M.D., Cunningham, R.L., Burclaff, J., Sibbel, G., Lo, H.G., Blanc, V., et al. (2018). Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis. EMBO J. 37, e98311.
Yousefi, M., Li, L., and Lengner, C.J. (2017). Hierarchy and plasticity in the intestinal stem cell compartment. Trends Cell Biol. 27, 753–764. Yousefi, M., Nakauka-Ddamba, A., Berry, C.T., Li, N., Schoenberger, J., Simeonov, K.P., Cedeno, R.J., Yu, Z., and Lengner, C.J. (2018). Calorie restriction governs intestinal epithelial regeneration through cell-autonomous regulation of mTORC1 in reserve stem cells. Stem Cell Reports 10, 703–711.
Basal Feet: Walking to the Discovery of a Novel Hybrid Cilium Rachael M. Fewell1 and Susan K. Dutcher2,* 1Department
of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA of Genetics, Washington University School of Medicine, St. Louis, MO, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2020.09.018 2Department
Cilia are important cell structures found on nearly all cells. In this issue of Developmental Cell, Mennella and colleagues investigate the molecular architecture of basal foot proteins in cells with primary or motile cilia and discover a hybrid cilium with a unique assembly that regulates polarity in multiciliated cells. Cilia are critical cell structures that move fluids or cells and can sense and respond to environmental signals. These tiny organelles project from the cell surface and have huge implications for cell function and thus disease. Currently, they are classified as either motile or primary cilia, based on structural differences (Paintrand et al., 1992). In the pair of articles by Mennella and colleagues in the current issue of Developmental Cell (Liu et al., 2020; Nguyen et al., 2020), the authors use proteomics, super-resolution microscopy, and state-of-the-art electron microscopy techniques to investigate the organization of an important structural component of cilia, the basal foot, to resolve differences between primary and motile cilia. Over the course of this structural and functional analysis, the authors discover a ‘‘hybrid’’ cilium with characteristics of both motile and primary cilia with a unique and novel function. This new hybrid cilium raises many questions about its role in regulating motile cilia. Present on nearly all cells, cilia play critical but diverse roles that vary with the cell and cilia type. Primary cilia act as environ-
mental sensing organelles and are membrane-encased single projections extending from the cell into the extracellular environment. Motile cilia are multiciliated, with rows of cilia that beat in a coordinated fashion. Motile cilia are restricted in distribution to regional subpopulations in the airway epithelial cells, the ependymal epithelium of the brain, and the fallopian tubes (Reiter and Leroux, 2017). In airway cells, the synchronized beating of motile cilia moves respiratory secretions for host defense (Spassky and Meunier, 2017). Both forms of cilia are anchored to the membrane via a basal body with basal ‘‘feet’’. In primary cilia there are nine basal feet, also known as subdistal appendages, that are needed for ciliogenesis (Figure 1C), whereas in motile cilia each of the hundreds of neighboring cilia per cell have just one basal foot per basal body that connects to cytoplasmic microtubules (Clare et al., 2014) (Figure 1B). A partial parts list of proteins that localize to the subdistal appendages (basal feet) are known from previous work (Kunimoto et al. 2012; Gupta et al. 2015). Using super-resolution and elec-
tron microscopy of retinal pigmented epithelial cells that have a single primary cilium, Mennella and colleagues (Liu et al., 2020; Nguyen et al., 2020) investigated the architecture of basal feet. Analysis defined three regions linked by coiled-coil protein assemblies and determined protein positions relative to the basal body microtubules (Figure 1A). The regions are named (I) the basal body anchoring region, (II) the scaffolding region, and (III) the microtubule anchoring region. Ninein, a well-known foot component, is found in region III and serves to connect regions II and III. The MTOC protein Centriolin (CNTRL) connects regions II and III and is one of six proteins proposed to be in region II. Loss of region II proteins in multiciliated cells results in the misorientation of the rows of cilia, lack of coordination among the motile cilia, and the loss of the apical microtubule network (Marshall and Kintner, 2008). Superresolution structured illumination microscopy (SIM) analysis showed that the basal foot of motile cilia had the same proteins, but their locations in the structure were slightly different.
Developmental Cell 55, October 26, 2020 ª 2020 Elsevier Inc. 115
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A
C
Region III Region II Region I
Figure 1. Diagram of Basal Feet (A) The basal foot in primary cilia has three regions connected by coiled-coil proteins (black lines). Region I is the basal-body-anchoring domain (red). Region II is the scaffolding region and contains ODF2, which has been shown to be critical for basal foot assembly as well as five other proteins (blue). Region III is the microtubule-anchoring domain and contains ninenin and CEP170 (green). (B) A single basal foot is found on motile cilia. (C) Multiple basal feet are found on primary cilia and the new hybrid cilium.
While investigating the composition of the basal foot in motile multiciliated cells, Mennella and his team found an unexpected arrangement in airway epithelial cells. While primary cilia have nine basal feet that appear in SIM images as a symmetrical ring, SIM images for motile cilia show just one focus associated with their lone basal foot. Among the hundreds of cilia in an airway epithelial cell, the authors were surprised to observe a single cilium that did not show the expected single focus but instead showed a ring of staining with region II antibodies, as is seen in a primary cilium. They used focused ion beam-scanning electron microscopy (FIB-SIM) to visualize this structure in more detail. They found a basal body with multiple basal feet that templates a beating cilium with central pair microtubules, nexin dynein regulatory complex, and radial spokes. They called it a ‘‘hybrid’’ cilium because it shares properties of both motile and primary cilia: the structures of a motile cilia but the basal feet pattern of a primary cilium. Another difference is that motile cilia have pillar microtubules (Clare et al., 2014), while the hybrid and primary cilia do not. The authors hypothesize this hybrid cilium has independent regulatory control from motile cilia, despite their colocalization. Accordingly, they examined the airway cells from patients with mutations in CCNO (a transcriptional regulator of ciliogenesis). Patients with mutations in CCNO have a primary ciliary dyskinesia phenotype, and their motile cilia cells 116 Developmental Cell 55, October 26, 2020
assemble only one or two cilia instead of the hundreds normally present (Lucas et al., 2020). The authors speculate that these one or two cilia are actually hybrid cilia and, using TEM analysis, show structurally they do indeed have multiple basal feet per basal body. They further investigate the regulatory mechanism underlying the formation of the hybrid cilia using the PLK4 inhibitor, centrinone. PLK4 is a kinase needed for duplication of centrioles, but not the amplification of basal bodies, in multiciliated cells (Wong et al., 2015). The authors show that treatment with centrinone causes reduction in the number of hybrid and primary cilia, but not in the number of motile cilia. This suggests the regulation of the hybrid cilia is distinct from motile cilia and more akin to that of primary cilia. The authors suggest the original mother centriole that is present before amplification of the basal bodies is perhaps templating this new hybrid cilium. The alignment of rows of cilia in a multiciliated cell requires fluid flow and then a ‘‘fine-tuning’’ of the placement of the basal bodies to generate directional mucociliary clearance. The basal feet link basal bodies together through their connection to the apical microtubule network (Marshall and Kintner, 2008). Mennella and his colleagues find that the hybrid cilium is positioned off center in cells, consistently to the back of the cell relative to the beating direction of the cilia. Interestingly, in the multiciliated cells of patients with dysfunctional immotile cilia, the hybrid cilium is
located more centrally. This finding suggests the positioning of the hybrid cilium may function to help align basal bodies and direct flow. Further corroborating this theory was their finding that treatment with centrinone, which causes reduction or loss of the hybrid cilium, resulted in a reduction in the alignment of the basal bodies. These findings raise many interesting questions. What are the regulators that transform the mother centriole in the airway into the hybrid cilium? PCD patients with altered basal body polarity may provide a source of interesting variants in these regulatory proteins. How is the position of the hybrid cilium achieved? Data in Liu et al. (2020) suggest that the flow generated by motile cilia plays a key role. It will be interesting to understand if the hybrid cilium is sensing a ligand or is responding to the mechanical forces. How does the hybrid cilium affect the positioning of the other 200 basal bodies? This last question is intriguing. How do this one basal body and its cilium signal the microtubule network and the other basal bodies to change their arrangement? This will be an important new area of research to begin to understand how this hybrid basal body/cilium functions in multiciliated cells. REFERENCES Clare, D.K., Magescas, J., Piolot, T., Dumoux, M., Vesque, C., Pichard, E., Dang, T., Duvauchelle, B., Poirier, F., and Delacour, D. (2014). Basal foot MTOC organizes pillar MTs required for coordination of beating cilia. Nat. Commun. 5, 4888. Gupta, G.D., Coyaud, E´., Gonc¸alves, J., Mojarad, B.A., Liu, Y., Wu, Q., Gheiratmand, L., Comartin, D., Tkach, J.M., Cheung, S.W., et al. (2015). A dynamic protein interaction landscape of the human centrosome-cilium interface. Cell 163, 1484–1499. Kunimoto, K., Yamazaki, Y., Nishida, T., Shinohara, K., Ishikawa, H., Hasegawa, T., Okanoue, T., Hamada, H., Noda, T., Tamura, A., et al. (2012). Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet. Cell 148, 189–200. Liu, Z., Nguyen, Q.P.H., Nanjundappa, R., Delgehyr, N., Megherbi, A., Doherty, R., Thompson, J., Jackson, C., Albulescu, A., Heng, Y.M., et al. (2020). Super-resolution microscopy and FIB-SEM imaging reveals parental centriole-derived, hybrid cilium in mammalian multiciliated cells. Dev. Cell 55, this issue, 224–236. Lucas, J.S., Davis, S.D., Omran, H., and Shoemark, A. (2020). Primary ciliary dyskinesia in
ll
Previews the genomics age. Lancet Respir. Med. 8, 202–216.
ture in primary and motile cilia. Dev. Cell 55, this issue, 209–223.
Marshall, W.F., and Kintner, C. (2008). Cilia orientation and the fluid mechanics of development. Curr. Opin. Cell Biol. 20, 48–52.
Paintrand, M., Moudjou, M., Delacroix, H., and Bornens, M. (1992). Centrosome organization and centriole architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107–128.
Nguyen, Q.P.H., Liu, Z., Albulescu, A., Ouyang, H., Zlock, L., Coyaud, E., Laurent, E., Finkbeiner, W., Moraes, T.J., Raught, B., and Vito Mennella, V. (2020). Comparative super-resolution mapping of basal feet reveals a modular but distinct architec-
Reiter, J.F., and Leroux, M.R. (2017). Genes and molecular pathways underpinning ciliopathies. Nat. Rev. Mol. Cell. Biol. 18, 533–547.
Spassky, N., and Meunier, A. (2017). The development and functions of multiciliated epithelia. 1198. Nat. Rev. Mol. Cell Biol. 18, 423–436.
Wong, Y.L., Anzola, J.V., Davis, R.L., Yoon, M., Motamedi, A., Kroll, A., Seo, C.P., Hsia, J.E., Kim, S.K., Mitchell, J.W., et al. (2015). Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155–1160.
Developmental Cell 55, October 26, 2020 117
Previews transdifferentiation are back in style. Nat. Rev. Mol. Cell Biol. 17, 413–425. Miao, Z.-F., Lewis, M.A., Cho, C.J., AdkinsThreats, M., Park, D., Brown, J.W., Sun, J.-X., Burclaff, J.R., Kennedy, S., Lu, J., et al. (2020). A dedicated evolutionarily conserved molecular network licenses differentiated cells to return to the cell cycle. Dev. Cell 55, this issue, 178–194. Rodgers, J.T., King, K.Y., Brett, J.O., Cromie, M.J., Charville, G.W., Maguire, K.K., Brunson, C.,
Mastey, N., Liu, L., Tsai, C.-R., et al. (2014). mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510, 393–396. Willet, S.G., Lewis, M.A., Miao, Z., Liu, D., Radyk, M.D., Cunningham, R.L., Burclaff, J., Sibbel, G., Lo, H.G., Blanc, V., et al. (2018). Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis. EMBO J. 37, e98311.
Yousefi, M., Li, L., and Lengner, C.J. (2017). Hierarchy and plasticity in the intestinal stem cell compartment. Trends Cell Biol. 27, 753–764. Yousefi, M., Nakauka-Ddamba, A., Berry, C.T., Li, N., Schoenberger, J., Simeonov, K.P., Cedeno, R.J., Yu, Z., and Lengner, C.J. (2018). Calorie restriction governs intestinal epithelial regeneration through cell-autonomous regulation of mTORC1 in reserve stem cells. Stem Cell Reports 10, 703–711.
Basal Feet: Walking to the Discovery of a Novel Hybrid Cilium Rachael M. Fewell1 and Susan K. Dutcher2,* 1Department
of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA of Genetics, Washington University School of Medicine, St. Louis, MO, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2020.09.018 2Department
Cilia are important cell structures found on nearly all cells. In this issue of Developmental Cell, Mennella and colleagues investigate the molecular architecture of basal foot proteins in cells with primary or motile cilia and discover a hybrid cilium with a unique assembly that regulates polarity in multiciliated cells. Cilia are critical cell structures that move fluids or cells and can sense and respond to environmental signals. These tiny organelles project from the cell surface and have huge implications for cell function and thus disease. Currently, they are classified as either motile or primary cilia, based on structural differences (Paintrand et al., 1992). In the pair of articles by Mennella and colleagues in the current issue of Developmental Cell (Liu et al., 2020; Nguyen et al., 2020), the authors use proteomics, super-resolution microscopy, and state-of-the-art electron microscopy techniques to investigate the organization of an important structural component of cilia, the basal foot, to resolve differences between primary and motile cilia. Over the course of this structural and functional analysis, the authors discover a ‘‘hybrid’’ cilium with characteristics of both motile and primary cilia with a unique and novel function. This new hybrid cilium raises many questions about its role in regulating motile cilia. Present on nearly all cells, cilia play critical but diverse roles that vary with the cell and cilia type. Primary cilia act as environ-
mental sensing organelles and are membrane-encased single projections extending from the cell into the extracellular environment. Motile cilia are multiciliated, with rows of cilia that beat in a coordinated fashion. Motile cilia are restricted in distribution to regional subpopulations in the airway epithelial cells, the ependymal epithelium of the brain, and the fallopian tubes (Reiter and Leroux, 2017). In airway cells, the synchronized beating of motile cilia moves respiratory secretions for host defense (Spassky and Meunier, 2017). Both forms of cilia are anchored to the membrane via a basal body with basal ‘‘feet’’. In primary cilia there are nine basal feet, also known as subdistal appendages, that are needed for ciliogenesis (Figure 1C), whereas in motile cilia each of the hundreds of neighboring cilia per cell have just one basal foot per basal body that connects to cytoplasmic microtubules (Clare et al., 2014) (Figure 1B). A partial parts list of proteins that localize to the subdistal appendages (basal feet) are known from previous work (Kunimoto et al. 2012; Gupta et al. 2015). Using super-resolution and elec-
tron microscopy of retinal pigmented epithelial cells that have a single primary cilium, Mennella and colleagues (Liu et al., 2020; Nguyen et al., 2020) investigated the architecture of basal feet. Analysis defined three regions linked by coiled-coil protein assemblies and determined protein positions relative to the basal body microtubules (Figure 1A). The regions are named (I) the basal body anchoring region, (II) the scaffolding region, and (III) the microtubule anchoring region. Ninein, a well-known foot component, is found in region III and serves to connect regions II and III. The MTOC protein Centriolin (CNTRL) connects regions II and III and is one of six proteins proposed to be in region II. Loss of region II proteins in multiciliated cells results in the misorientation of the rows of cilia, lack of coordination among the motile cilia, and the loss of the apical microtubule network (Marshall and Kintner, 2008). Superresolution structured illumination microscopy (SIM) analysis showed that the basal foot of motile cilia had the same proteins, but their locations in the structure were slightly different.
Developmental Cell 55, October 26, 2020 ª 2020 Elsevier Inc. 115
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Previews B
A
C
Region III Region II Region I
Figure 1. Diagram of Basal Feet (A) The basal foot in primary cilia has three regions connected by coiled-coil proteins (black lines). Region I is the basal-body-anchoring domain (red). Region II is the scaffolding region and contains ODF2, which has been shown to be critical for basal foot assembly as well as five other proteins (blue). Region III is the microtubule-anchoring domain and contains ninenin and CEP170 (green). (B) A single basal foot is found on motile cilia. (C) Multiple basal feet are found on primary cilia and the new hybrid cilium.
While investigating the composition of the basal foot in motile multiciliated cells, Mennella and his team found an unexpected arrangement in airway epithelial cells. While primary cilia have nine basal feet that appear in SIM images as a symmetrical ring, SIM images for motile cilia show just one focus associated with their lone basal foot. Among the hundreds of cilia in an airway epithelial cell, the authors were surprised to observe a single cilium that did not show the expected single focus but instead showed a ring of staining with region II antibodies, as is seen in a primary cilium. They used focused ion beam-scanning electron microscopy (FIB-SIM) to visualize this structure in more detail. They found a basal body with multiple basal feet that templates a beating cilium with central pair microtubules, nexin dynein regulatory complex, and radial spokes. They called it a ‘‘hybrid’’ cilium because it shares properties of both motile and primary cilia: the structures of a motile cilia but the basal feet pattern of a primary cilium. Another difference is that motile cilia have pillar microtubules (Clare et al., 2014), while the hybrid and primary cilia do not. The authors hypothesize this hybrid cilium has independent regulatory control from motile cilia, despite their colocalization. Accordingly, they examined the airway cells from patients with mutations in CCNO (a transcriptional regulator of ciliogenesis). Patients with mutations in CCNO have a primary ciliary dyskinesia phenotype, and their motile cilia cells 116 Developmental Cell 55, October 26, 2020
assemble only one or two cilia instead of the hundreds normally present (Lucas et al., 2020). The authors speculate that these one or two cilia are actually hybrid cilia and, using TEM analysis, show structurally they do indeed have multiple basal feet per basal body. They further investigate the regulatory mechanism underlying the formation of the hybrid cilia using the PLK4 inhibitor, centrinone. PLK4 is a kinase needed for duplication of centrioles, but not the amplification of basal bodies, in multiciliated cells (Wong et al., 2015). The authors show that treatment with centrinone causes reduction in the number of hybrid and primary cilia, but not in the number of motile cilia. This suggests the regulation of the hybrid cilia is distinct from motile cilia and more akin to that of primary cilia. The authors suggest the original mother centriole that is present before amplification of the basal bodies is perhaps templating this new hybrid cilium. The alignment of rows of cilia in a multiciliated cell requires fluid flow and then a ‘‘fine-tuning’’ of the placement of the basal bodies to generate directional mucociliary clearance. The basal feet link basal bodies together through their connection to the apical microtubule network (Marshall and Kintner, 2008). Mennella and his colleagues find that the hybrid cilium is positioned off center in cells, consistently to the back of the cell relative to the beating direction of the cilia. Interestingly, in the multiciliated cells of patients with dysfunctional immotile cilia, the hybrid cilium is
located more centrally. This finding suggests the positioning of the hybrid cilium may function to help align basal bodies and direct flow. Further corroborating this theory was their finding that treatment with centrinone, which causes reduction or loss of the hybrid cilium, resulted in a reduction in the alignment of the basal bodies. These findings raise many interesting questions. What are the regulators that transform the mother centriole in the airway into the hybrid cilium? PCD patients with altered basal body polarity may provide a source of interesting variants in these regulatory proteins. How is the position of the hybrid cilium achieved? Data in Liu et al. (2020) suggest that the flow generated by motile cilia plays a key role. It will be interesting to understand if the hybrid cilium is sensing a ligand or is responding to the mechanical forces. How does the hybrid cilium affect the positioning of the other 200 basal bodies? This last question is intriguing. How do this one basal body and its cilium signal the microtubule network and the other basal bodies to change their arrangement? This will be an important new area of research to begin to understand how this hybrid basal body/cilium functions in multiciliated cells. REFERENCES Clare, D.K., Magescas, J., Piolot, T., Dumoux, M., Vesque, C., Pichard, E., Dang, T., Duvauchelle, B., Poirier, F., and Delacour, D. (2014). Basal foot MTOC organizes pillar MTs required for coordination of beating cilia. Nat. Commun. 5, 4888. Gupta, G.D., Coyaud, E´., Gonc¸alves, J., Mojarad, B.A., Liu, Y., Wu, Q., Gheiratmand, L., Comartin, D., Tkach, J.M., Cheung, S.W., et al. (2015). A dynamic protein interaction landscape of the human centrosome-cilium interface. Cell 163, 1484–1499. Kunimoto, K., Yamazaki, Y., Nishida, T., Shinohara, K., Ishikawa, H., Hasegawa, T., Okanoue, T., Hamada, H., Noda, T., Tamura, A., et al. (2012). Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet. Cell 148, 189–200. Liu, Z., Nguyen, Q.P.H., Nanjundappa, R., Delgehyr, N., Megherbi, A., Doherty, R., Thompson, J., Jackson, C., Albulescu, A., Heng, Y.M., et al. (2020). Super-resolution microscopy and FIB-SEM imaging reveals parental centriole-derived, hybrid cilium in mammalian multiciliated cells. Dev. Cell 55, this issue, 224–236. Lucas, J.S., Davis, S.D., Omran, H., and Shoemark, A. (2020). Primary ciliary dyskinesia in
ll
Previews the genomics age. Lancet Respir. Med. 8, 202–216.
ture in primary and motile cilia. Dev. Cell 55, this issue, 209–223.
Marshall, W.F., and Kintner, C. (2008). Cilia orientation and the fluid mechanics of development. Curr. Opin. Cell Biol. 20, 48–52.
Paintrand, M., Moudjou, M., Delacroix, H., and Bornens, M. (1992). Centrosome organization and centriole architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107–128.
Nguyen, Q.P.H., Liu, Z., Albulescu, A., Ouyang, H., Zlock, L., Coyaud, E., Laurent, E., Finkbeiner, W., Moraes, T.J., Raught, B., and Vito Mennella, V. (2020). Comparative super-resolution mapping of basal feet reveals a modular but distinct architec-
Reiter, J.F., and Leroux, M.R. (2017). Genes and molecular pathways underpinning ciliopathies. Nat. Rev. Mol. Cell. Biol. 18, 533–547.
Spassky, N., and Meunier, A. (2017). The development and functions of multiciliated epithelia. 1198. Nat. Rev. Mol. Cell Biol. 18, 423–436.
Wong, Y.L., Anzola, J.V., Davis, R.L., Yoon, M., Motamedi, A., Kroll, A., Seo, C.P., Hsia, J.E., Kim, S.K., Mitchell, J.W., et al. (2015). Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155–1160.
Developmental Cell 55, October 26, 2020 117