A Chlamydomonas mutant, vfl2, displays aberrant flagellar numbers due to an E101K mutation in the centrin gene. We isolated 21 revertants–12 intragenic and 9 extragenic–that suppress the mutant's abnormal phenotype. AlphaFold2 modeling indicates that the wild-type, mutant, and revertant proteins are structurally similar. However, the vfl2 mutant centrin forms three salt bridges in the central region of the core a-helix, whereas wild-type and all intragenic revertants have only two salt bridges or disrupted helix continuity. These results indicate that flexibility of the central helix, maintained by limited electrostatic interactions, is important for proper centrin function.
","acknowledgements":"We thank Ms. and Mr. Takeru Ando, Miku Arai, Shota Umeki, Tomoya Oshima, and Atsuhiro Hattori (Chuo University) for their assistance with sequencing analysis. We also thank Dr. Akira Noga (Chuo University) for his critical reading of the manuscript and helpful suggestions.
","authors":[{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","fundingAcquisition","dataCuration","writing_originalDraft"],"email":"minoura.87b@g.chuo-u.ac.jp","firstName":"Takako","lastName":"Kato-Minoura","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4033-2427"},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Japan"],"departments":[""],"credit":["investigation"],"email":"a20.tbkx@g.chuo-u.ac.jp","firstName":"Ichiro","lastName":"Sasaki","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Japan"],"departments":[""],"credit":["conceptualization","supervision"],"email":"ritsu.kamiya@gmail.com","firstName":"Ritsu","lastName":"Kamiya","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Sanger sequence data of vfl2 and 21 revertants. Only partial regions, bases 286 to 306, where the A at the start codon is 1, are shown. The red arrows indicate the vfl2-type SNP that results in the E101K mutation. The blue arrows indicate additional changes, grouped by revertant type.
","doi":null,"resourceType":"Image","name":"sup_fig_1(288).jpg","url":"https://portal.micropublication.org/uploads/c94ff44d521d0dc5efd1abdb4265d960.jpg"}],"funding":"This study was supported in part by the Chuo University Grant for Special Research.
","image":{"url":"https://portal.micropublication.org/uploads/3e6ffeb49bb5aaf9990f72bf48a81445.jpg"},"imageCaption":"A, Genotypes of the vfl2 revertants. B, wild-type overall centrin (Vfl2) structure (PDB: 3QRX, Sosa et al., 2011); C, magnified mid portion of wild-type centrin (box region in B) D-J, AlphaFold2-predicted centrin structures in vfl2 (D), vfl2-R201 (E), vfl2-R206 (F), vfl2-R214/R221 (G), vfl2-R220/R222 (H), vfl2-R202/R205/R217 (I), vfl2-R205 (J). In B, Orange spheres indicate Ca2+ ions. Light green, dark green, light teal, and deep teal indicate the 1st to 4th EF-hand motifs, respectively. In the central a-helices in B-J, acidic/basic/mutated amino acids are shown as a stick model, where acidic amino acids (E or D) are colored red, basic (K or R) are colored blue, and other mutated amino acids are colored orange; in C-J, the distances of the salt bridges are indicated in Å. K: alignments of the amino acid sequences of the mid portion of the central a-helix in wild type, vfl2, and type 2 and 3 revertants.
","imageTitle":"Summary of vfl2 revertant classes and associated mutations
","methods":"Revertant isolation
The mutant vfl2 (Kuchka & Jarvik, 1982) was cultured in liquid TAP medium (Gorman & Levine, 1965) for 3 days, placed in a Petri dish, irradiated with a UV lamp (20 W) from 40 cm above for 2 min, and transferred to test tubes with 8 mL liquid TAP. After being kept in the dark for 1 day to prevent photo-recovery, the upper 0.5 mL of each test tube, containing swimming cells, was transferred to another test tube with 8 mL of fresh medium. This process was repeated a few times. Finally, the cells swimming on the upper surface of each test-tube culture were inoculated onto TAP agar plates to produce single colonies. Each clone was cultured in a multi-well plate with TAP medium for motility assessment under an inverted microscope. Strains that showed near-wild-type motility were saved as revertants.
Genomic DNA sequencing
Genomic DNA from vfl2 and its revertants was purified using the method of Rochaix et al. (1988), with some modifications. The coding region was then amplified using primers 5vfl2-F and 3vfl2-R (see Reagents/Strains). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and their sequences were determined using primers 5vfl2-F and 5vfl2-F2 (see Reagents/Strains). Sequencing was entrusted to Macrogen Japan.
3D structure prediction
AlphaFold2 (Jumper et al., 2021) was used to predict the 3D structure of vfl2 and revertants centrins, then plotted in PyMOL (DeLano Scientific,https://www.pymol.org).
","reagents":"PRIMER NAME | SEQUENCE |
5vfl2-F | 5'-AGTCTGCTGCTGGAGCTCAGACC-3 |
3vfl2-R | 5'-AATTTATTATGTGCCGTGCTGTGCAC-3' |
5vfl2-F2 | 5'-TGTGGGCATAGCGGTGACAAA-3' |
STRAIN | GENOTYPE | AVAIRABLE FROM |
CC-125 | Wild type | Chlamydomonas Resource Center |
vfl2 | E101K in Vfl2 | Chlamydomonas Resource Center (Kuchka & Jarvik, 1982; Taillon et al., 1992) |
vfl2-R201~R222 | see Figure 1A. | All revertants isolated in this study will be deposited in the Chlamydomonas Resource Center. |
Centrin is a highly conserved eukaryotic protein of ~ 20 kDa with four Ca2+-binding EF-hand motifs. In animal cells, centrin localizes to the centriole, the core structure of the centrosome, and plays essential roles in mitotic spindle formation and the organization of cytoplasmic microtubules. In ciliated cells, the centriole becomes a basal body and functions as the template for axonemal assembly. Centrin is thus essential for both cell division and ciliation (flagellation).
In the unicellular biflagellate green alga Chlamydomonas, centrin is expressed from the gene vfl2. It mainly localizes to three distinct sites in interphase cells: fibers connecting the nuclei and basal body, distal striated fibers (DSF) that connect the two basal bodies, and the transition zone located between the basal body and the flagellum (Wright et al., 1985; Schulze et al., 1987; Salisbury et al., 1988; Sanders & Salisbury, 1989). In most cases, centrin is contained in fibrous structures, which can contract at higher Ca2+ concentrations (Wingfield and Lechtreck, 2018). Centrin in the transition zone has been suggested to participate in flagellar shedding (Sanders and Salisbury, 1989), and the DSF centrin to function in changing the angle between the two flagella in a Ca2+-dependent manner (Salisbury et al., 1987; Hayashi et al., 1998).
The Chlamydomonas mutant vfl2, which displays aberrant flagellar numbers, carries centrin-gene mutation causing a Glu-to-Lys substitution at position 101 (Kuchka & Jarvik, 1982; Taillon et al., 1992). Taillon et al. (1992) mutagenized this mutant and obtained 19 revertants that apparently recovered wild-type flagellation. Ten of these had changes at Lys101 to one of five amino acids (Taillon et al., 1992). The present study extended previous work by isolating 21 additional phenotypic revertants and identifying several novel amino acid changes (Figure 1A). All revertants displayed almost normal swimming phenotypes due to recovery of the flagellar-number phenotype. Sequence analysis revealed four classes: type 1, wild-type sequence restored; type 2, Lys101 replaced by another amino acid except glutamic acid; type 3, additional mutation(s) present while the original E101K mutation was retained; and type 4, no mutation found except the original E101K. Types 1 to 3 are intragenic revertants, whereas type 4 likely reflects extragenic suppression. Some classes correspond to those previously reported, while others represent newly identified revertant types.
We focus here on type 2 and type 3 mutants with intragenic changes, although type 4 mutants are also interesting and warrant future studies, as these revertants indicate mutations in non-centrin proteins. As shown in Figure 1E-J, all amino acid changes in type 2 and type 3 occurred in the central portion of the central a-helix in centrin. Within this region, electrostatic interactions occur between different amino acid side chains. For example, in the two pitches of the a-helix N-terminal to position E101, attractive interactions occur between E95 and R99, and between R96 and E100, while a repulsive interaction occurs between D97 and E101 (Figure 1B, C). In contrast, in vfl2 centrin, there are three attractively interacting amino-acid pairs: E95-R99, R96-E100, and D97-K101 (Figure 1D). This suggests that the α-helix around position 101 is more stabilized in the vfl2 mutant than in the wild type, although AlphaFold2 (Jumper et al., 2021) predicts similar structures for wild-type and vfl2 centrins. All type 2 and type 3 revertants isolated in this study have either only two attractive amino-acid pairs or a proline kink in this region (Figure 1E-J). In other words, no revertants were isolated that retained the three pairs of oppositely charged amino acids present in the vfl2 mutant. These results suggest that appropriate centrin flexibility–arising from limited electrostatic interaction or disruption of helix continuity–is important for centrin function at the flagellar base.
Based on sequence analyses of revertants, we propose that excessive stabilization of the centrin a-helix around position 101 is detrimental to its function, and that some flexibility in this region may be necessary. This idea contrasts with the proposal of Taillon et al. (1992) that E101 stabilizes the centrin structure by forming a salt bridge with another basic amino acid and that this stabilization is important for centrin function. In calmodulin (CaM), a homolog of centrin, the middle region of the corresponding central helix functions as a \"flexible tether\" crucial to its function (Persechini et al., 1988a). In CaM, the central region is almost entirely composed of negatively charged amino acids and therefore barely forms an a-helix (Persechini et al., 1988b; Barbato et al., 1992, for review, see Nelson and Chazin, 1998). Thus, some flexibility in the central region of the central helix may also be important for centrin. Having only two salt bridges in centrin's central helix may therefore be crucial. (Expended Data: Supplementary Figure 1)
","references":[{"reference":"Adams GM, Wright RL, Jarvik JW. 1985. Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.. The Journal of cell biology 100: 955-964.
","pubmedId":"","doi":"10.1083/jcb.100.3.955"},{"reference":"Barbato G, Ikura M, Kay LE, Pastor RW, Bax A. 1992. Backbone dynamics of calmodulin studied by nitrogen-15 relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31: 5269-5278.
","pubmedId":"","doi":"10.1021/bi00138a005"},{"reference":"Gorman DS, Levine RP. 1965. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.. Proceedings of the National Academy of Sciences 54: 1665-1669.
","pubmedId":"","doi":"10.1073/pnas.54.6.1665"},{"reference":"Hayashi M, Yagi T, Yoshimura K, Kamiya R. 1998. Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas. Cell Motil Cytoskeleton 41(1): 49-56.
","pubmedId":"9744298","doi":""},{"reference":"Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al., Hassabis. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589.
","pubmedId":"","doi":"10.1038/s41586-021-03819-2"},{"reference":"Kuchka MR, Jarvik JW. 1982. Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella.. The Journal of cell biology 92: 170-175.
","pubmedId":"","doi":"10.1083/jcb.92.1.170"},{"reference":"Melkonian M. 1978. Structure and significance of cruciate flagellar root systems in green algae: Comparative investigations in species ofChlorosarcinopsis (Chlorosarcinales). Plant Systematics and Evolution 130: 265-292.
","pubmedId":"","doi":"10.1007/BF00982810"},{"reference":"Nelson MR, Chazin WJ. 1998. An interaction‐based analysis of calcium‐induced conformational changes in Ca2+ sensor proteins. Protein Science 7: 270-282.
","pubmedId":"","doi":"10.1002/pro.5560070206"},{"reference":"Persechini A, Kretsinger RH. 1988. The central helix of calmodulin functions as a flexible tether. J Biol Chem 263(25): 12175-8.
","pubmedId":"3137220","doi":""},{"reference":"Persechini A, Kretsinger RH. 1988. Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function. J Cardiovasc Pharmacol 12 Suppl 5: S1-12.
","pubmedId":"2469863","doi":""},{"reference":"Rochaix JD, Mayfield S, Goldschmidt CM, Erickson J. 1988. Molecular biology of Chlamydomonas. In: Shaw CH (ed) Plant molecular biology, a practical approach. IRL Press, Oxford, pp 253–275.
","pubmedId":"","doi":""},{"reference":"Salisbury JL, Baron AT, Sanders MA. 1988. The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.. The Journal of cell biology 107: 635-641.
","pubmedId":"","doi":"10.1083/jcb.107.2.635"},{"reference":"Salisbury JL, Sanders MA, Harpst L. 1987. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii.. The Journal of cell biology 105: 1799-1805.
","pubmedId":"","doi":"10.1083/jcb.105.4.1799"},{"reference":"Sanders MA, Salisbury JL. 1989. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.. The Journal of cell biology 108: 1751-1760.
","pubmedId":"","doi":"10.1083/jcb.108.5.1751"},{"reference":"Schulze D, Robenek H, McFadden GI, Melkonian M. 1987. Immunolocalization of a Ca2+-modulated contractile protein in the flagellar apparatus of green algae: the nucleus-basal body connector. Eur. J. Cell Biol. 45: 51–61.
","pubmedId":"","doi":""},{"reference":"Sosa LdV, Alfaro E, Santiago J, Narváez D, Rosado MC, Rodríguez A, et al., Pastrana‐Ríos. 2011. The structure, molecular dynamics, and energetics of centrin–melittin complex. Proteins: Structure, Function, and Bioinformatics 79: 3132-3143.
","pubmedId":"","doi":"10.1002/prot.23142"},{"reference":"Taillon BE, Adler SA, Suhan JP, Jarvik JW. 1992. Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.. The Journal of cell biology 119: 1613-1624.
","pubmedId":"","doi":"10.1083/jcb.119.6.1613"},{"reference":"Wingfield JL, Lechtreck KF. 2018. Chlamydomonas Basal Bodies as Flagella Organizing Centers. Cells 7: 79.
","pubmedId":"","doi":"10.3390/cells7070079"},{"reference":"Wright RL, Salisbury J, Jarvik JW. 1985. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.. The Journal of cell biology 101: 1903-1912.
","pubmedId":"","doi":"10.1083/jcb.101.5.1903"}],"title":"Novel revertants of Chlamydomonas vfl2 suggest a role for a flexible central a-helical region in centrin function in flagellar basal bodies
","reviews":[{"reviewer":{"displayName":"Gregory J Pazour"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"436fd3c1-893b-46d1-bb2a-73e92e9e9d60","decision":"edit","abstract":"A Chlamydomonas mutant, vfl2, displays aberrant flagellar numbers due to an E101K mutation in the centrin gene. We isolated 21 revertants–12 intragenic and 9 extragenic–that suppress the mutant's abnormal phenotype. AlphaFold2 modeling indicates that the wild-type, mutant, and revertant proteins are structurally similar. However, the vfl2 mutant centrin forms three salt bridges in the central region of the core α-helix, whereas the wild type and all intragenic revertants have only two salt bridges or disrupted helix continuity. These results indicate that flexibility of the central helix, maintained by limited electrostatic interactions, is important for proper centrin function.
","acknowledgements":"We thank Ms. and Mr. Takeru Ando, Miku Arai, Shota Umeki, Tomoya Oshima, and Atsuhiro Hattori (Chuo University) for their assistance with sequencing analysis. We also thank Dr. Akira Noga (Chuo University) for his critical reading of the manuscript and helpful suggestions.
","authors":[{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","fundingAcquisition","dataCuration","writing_originalDraft"],"email":"minoura.87b@g.chuo-u.ac.jp","firstName":"Takako","lastName":"Kato-Minoura","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4033-2427"},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["investigation"],"email":"i.chan.ex2017@gmail.com","firstName":"Ichiro","lastName":"Sasaki","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","supervision"],"email":"ritsu.kamiya@gmail.com","firstName":"Ritsu","lastName":"Kamiya","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Sanger sequencing data of vfl2 and 21 revertants. Only partial regions, bases 286 to 306, where the A at the start codon is 1, are shown. The red arrows indicate the vfl2-type SNP that results in the E101K mutation. The blue arrows indicate additional changes, grouped by revertant type.
","doi":null,"resourceType":"Image","name":"sup_fig_1(288).jpg","url":"https://portal.micropublication.org/uploads/c94ff44d521d0dc5efd1abdb4265d960.jpg"}],"funding":"This study was supported in part by the Chuo University Grant for Special Research.
","image":{"url":"https://portal.micropublication.org/uploads/28723abb348afeddc353c77dbb41487f.jpg"},"imageCaption":"A, Genotypes of the vfl2 revertants. B, wild-type overall centrin (Vfl2) structure (PDB: 3QRX, Sosa et al., 2011); C, magnified mid portion of wild-type centrin (box region in B); D–J, AlphaFold2-predicted centrin structures in vfl2 (D), vfl2-R201 (E), vfl2-R206 (F), vfl2-R214/R221 (G), vfl2-R220/R222 (H), vfl2-R202/R205/R217 (I), vfl2-R205 (J). In B, orange spheres indicate Ca2+ ions. Light green, dark green, light teal, and deep teal indicate the 1st to 4th EF-hand motifs, respectively. In the central α-helices in B–J, acidic/basic amino acids are shown as a stick model, with acidic amino acids (E or D) colored red and basic amino acids (K or R) colored blue. Other mutated amino acids are colored orange. In C–J, the salt bridge distances are indicated in Å, and the specific amino acid variations are shown at the bottom of each panel. The numbers in the schematic diagram of panel C indicate the amino acid positions in the wild-type primary sequence, illustrating their spatial arrangement along the pitches of the α-helix. Note that the amino acid at position 98 is not shown except in panel I, as this residue is located on the back side of the helix and not visible in the ribbon diagrams. K: alignments of the amino acid sequences of the mid portion of the central α-helix in wild type, vfl2, and type 2 and type 3 revertants.
","imageTitle":"Summary of vfl2 revertant classes and associated mutations
","methods":"Revertant isolation
The mutant vfl2 (Kuchka & Jarvik, 1982) was cultured in liquid TAP medium (Gorman & Levine, 1965) for 3 days, placed in a Petri dish, irradiated with a UV lamp (20 W) from 40 cm above for 2 min, and transferred to test tubes with 8 mL liquid TAP. After being kept in the dark for 1 day to prevent photo-recovery, the upper 0.5 mL of each test tube, containing swimming cells, was transferred to another test tube with 8 mL of fresh medium. This process was repeated a few times. Finally, the cells swimming on the upper surface of each test-tube culture were inoculated onto TAP agar plates to produce single colonies. Each clone was cultured in a multi-well plate with TAP medium for motility assessment under an inverted microscope. Strains that showed near-wild-type motility were saved as revertants.
Genomic DNA sequencing
Genomic DNA from vfl2 and its revertants was purified using the method of Rochaix et al. (1988), with some modifications. The coding region was then amplified using primers 5vfl2-F and 3vfl2-R (see Reagents/Strains). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and their sequences were determined using primers 5vfl2-F and 5vfl2-F2 (see Reagents/Strains). Sequencing was entrusted to Macrogen Japan.
3D structure prediction
AlphaFold2 (Jumper et al., 2021) was used to predict the 3D structure of vfl2 and revertants centrins, then plotted in PyMOL (DeLano Scientific,https://www.pymol.org).
","reagents":"PRIMER NAME | SEQUENCE |
5vfl2-F | 5'-AGTCTGCTGCTGGAGCTCAGACC-3 |
3vfl2-R | 5'-AATTTATTATGTGCCGTGCTGTGCAC-3' |
5vfl2-F2 | 5'-TGTGGGCATAGCGGTGACAAA-3' |
STRAIN | GENOTYPE | AVAIRABLE FROM |
CC-125 | Wild type | Chlamydomonas Resource Center |
vfl2 | E101K in Vfl2 | Chlamydomonas Resource Center (Kuchka & Jarvik, 1982; Taillon et al., 1992) |
vfl2-R201~R222 | see Figure 1A. | All revertants isolated in this study will be deposited in the Chlamydomonas Resource Center. |
Centrin is a highly conserved eukaryotic protein of ~ 20 kDa with four Ca2+-binding EF-hand motifs. In animal cells, centrin localizes to the centriole, the core structure of the centrosome, and plays essential roles in mitotic spindle formation and cytoplasmic microtubule organization. In ciliated cells, the centriole becomes a basal body and functions as the template for axonemal assembly. Centrin is thus essential for both cell division and ciliation (flagellation).
In the unicellular biflagellate green alga Chlamydomonas, centrin is expressed from the gene vfl2. It mainly localizes to three distinct sites in interphase cells: fibers connecting the nuclei and basal bodies, distal striated fibers (DSF) that connect the two basal bodies, and the transition zone located between the basal body and the flagellum (Wright et al., 1985; Schulze et al., 1987; Salisbury et al., 1988; Sanders & Salisbury, 1989). In most cases, centrin is contained in fibrous structures, which can contract at higher Ca2+ concentrations (Wingfield and Lechtreck, 2018). Centrin in the transition zone has been suggested to participate in flagellar shedding (Sanders and Salisbury, 1989), and the DSF centrin to function in changing the angle between the two flagella in a Ca2+-dependent manner (Salisbury et al., 1987; Hayashi et al., 1998).
The Chlamydomonas mutant vfl2, which displays aberrant flagellar numbers, carries a centrin-gene mutation causing a Glu-to-Lys substitution at position 101 (Kuchka & Jarvik, 1982; Taillon et al., 1992). Taillon et al. (1992) mutagenized this mutant and obtained 19 revertants that apparently recovered wild-type flagellation. Ten of these had changes at Lys101 to one of five amino acids (Taillon et al., 1992). The present study extended previous work by isolating 21 additional phenotypic revertants and identifying several novel amino acid changes (Figure 1A). All revertants displayed near-normal swimming phenotypes due to recovery of normal flagellar number. Sequence analysis revealed four classes: type 1, in which the wild-type sequence was restored; type 2, in which Lys101 was replaced by an amino acid other than the original glutamic acid; type 3, which harbored additional mutation(s) while retaining the original E101K mutation; and type 4, in which no mutation was found other than E101K. Types 1 through 3 are intragenic revertants, whereas type 4 likely reflects extragenic suppression. Some classes match those previously reported, whereas others represent newly identified revertant types.
We focus here on type 2 and type 3 mutants with intragenic changes, although type 4 mutants are also interesting and warrant future studies, as these revertants indicate mutations in non-centrin proteins. As shown in Figure 1E—J, all amino acid changes in type 2 and type 3 occurred in the central portion of the central α-helix in centrin. Within this region, electrostatic interactions occur between different amino acid side chains. For example, in the two pitches of the α-helix N-terminal to position E101, attractive interactions occur between E95 and R99, and between R96 and E100, while a repulsive interaction occurs between D97 and E101 (Figure 1B, C). In contrast, in vfl2 centrin, there are three attractively interacting amino-acid pairs: E95-R99, R96-E100, and D97-K101 (Figure 1D). This suggests that the α-helix around position 101 is more stabilized in vfl2 than in wild type, although AlphaFold2 (Jumper et al., 2021) predicts similar structures for wild-type and vfl2 centrins. All type 2 and type 3 revertants isolated in this study have either only two attractive amino-acid pairs or a proline kink in this region (Figure 1E—J). In other words, no revertants were isolated that retained the three pairs of oppositely charged amino acids present in vfl2. These results suggest that appropriate centrin flexibility—arising from limited electrostatic interaction or disruption of helix continuity—is important for centrin function at the flagellar base.
Notably, among the four types of amino acid substitutions at position 101 reported by Taillon et al. (1992)—specifically I, M, T, and N—our study isolated all but E101N. Although we did not isolate this mutation in our screen, an E101N substitution does not introduce a basic side chain and therefore cannot form the third salt bridge with D97, leaving the central helix with only two salt bridges. Thus, all revertants obtained by Taillon et al. (1992) likely have centrins with a more flexible central helix than the original vfl2 centrin, consistent with our proposed model.
Furthermore, Taillon et al. (1992) reported structural deficiencies in the DSF in some of their revertants. Although we have not examined the DSF structures in our isolated revertants, they may harbor similar defects. How central-helix flexibility specifically affects centrin function in regulating basal body number and DSF structure remains an important subject for future studies.
Based on sequence analyses of revertants, we propose that excessive stabilization of the centrin α-helix around position 101 is detrimental to its function, and that some flexibility in this region may be necessary. This idea contrasts with the proposal of Taillon et al. (1992) that E101 stabilizes the centrin structure by forming a salt bridge with another basic amino acid and that this stabilization is important for centrin function. In calmodulin (CaM), a homolog of centrin, the middle region of the corresponding central helix functions as a \"flexible tether\" crucial to its function (Persechini et al., 1988a). In CaM, the central region is almost entirely composed of negatively charged amino acids and therefore barely forms an α-helix (Persechini et al., 1988b; Barbato et al., 1992, for review, see Nelson and Chazin, 1998). Thus, some flexibility in the central region of the central helix may also be essential for centrin function. Having only two salt bridges in centrin's central helix may therefore be important for allowing this flexibility; this could be tested by generating mutants with additional salt bridges near position 101 in future studies. (Extended Data: Supplementary Figure 1)
","references":[{"reference":"Adams GM, Wright RL, Jarvik JW. 1985. Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.. The Journal of cell biology 100: 955-964.
","pubmedId":"","doi":"10.1083/jcb.100.3.955"},{"reference":"Barbato G, Ikura M, Kay LE, Pastor RW, Bax A. 1992. Backbone dynamics of calmodulin studied by nitrogen-15 relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31: 5269-5278.
","pubmedId":"","doi":"10.1021/bi00138a005"},{"reference":"Gorman DS, Levine RP. 1965. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.. Proceedings of the National Academy of Sciences 54: 1665-1669.
","pubmedId":"","doi":"10.1073/pnas.54.6.1665"},{"reference":"Hayashi M, Yagi T, Yoshimura K, Kamiya R. 1998. Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas. Cell Motil Cytoskeleton 41(1): 49-56.
","pubmedId":"9744298","doi":""},{"reference":"Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al., Hassabis. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589.
","pubmedId":"","doi":"10.1038/s41586-021-03819-2"},{"reference":"Kuchka MR, Jarvik JW. 1982. Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella.. The Journal of cell biology 92: 170-175.
","pubmedId":"","doi":"10.1083/jcb.92.1.170"},{"reference":"Melkonian M. 1978. Structure and significance of cruciate flagellar root systems in green algae: Comparative investigations in species ofChlorosarcinopsis (Chlorosarcinales). Plant Systematics and Evolution 130: 265-292.
","pubmedId":"","doi":"10.1007/BF00982810"},{"reference":"Nelson MR, Chazin WJ. 1998. An interaction‐based analysis of calcium‐induced conformational changes in Ca2+ sensor proteins. Protein Science 7: 270-282.
","pubmedId":"","doi":"10.1002/pro.5560070206"},{"reference":"Persechini A, Kretsinger RH. 1988. The central helix of calmodulin functions as a flexible tether. J Biol Chem 263(25): 12175-8.
","pubmedId":"3137220","doi":""},{"reference":"Persechini A, Kretsinger RH. 1988. Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function. J Cardiovasc Pharmacol 12 Suppl 5: S1-12.
","pubmedId":"2469863","doi":""},{"reference":"Rochaix JD, Mayfield S, Goldschmidt CM, Erickson J. 1988. Molecular biology of Chlamydomonas. In: Shaw CH (ed) Plant molecular biology, a practical approach. IRL Press, Oxford, pp 253–275.
","pubmedId":"","doi":""},{"reference":"Salisbury JL, Baron AT, Sanders MA. 1988. The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.. The Journal of cell biology 107: 635-641.
","pubmedId":"","doi":"10.1083/jcb.107.2.635"},{"reference":"Salisbury JL, Sanders MA, Harpst L. 1987. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii.. The Journal of cell biology 105: 1799-1805.
","pubmedId":"","doi":"10.1083/jcb.105.4.1799"},{"reference":"Sanders MA, Salisbury JL. 1989. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.. The Journal of cell biology 108: 1751-1760.
","pubmedId":"","doi":"10.1083/jcb.108.5.1751"},{"reference":"Schulze D, Robenek H, McFadden GI, Melkonian M. 1987. Immunolocalization of a Ca2+-modulated contractile protein in the flagellar apparatus of green algae: the nucleus-basal body connector. Eur. J. Cell Biol. 45: 51–61.
","pubmedId":"","doi":""},{"reference":"Sosa LdV, Alfaro E, Santiago J, Narváez D, Rosado MC, Rodríguez A, et al., Pastrana‐Ríos. 2011. The structure, molecular dynamics, and energetics of centrin–melittin complex. Proteins: Structure, Function, and Bioinformatics 79: 3132-3143.
","pubmedId":"","doi":"10.1002/prot.23142"},{"reference":"Taillon BE, Adler SA, Suhan JP, Jarvik JW. 1992. Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.. The Journal of cell biology 119: 1613-1624.
","pubmedId":"","doi":"10.1083/jcb.119.6.1613"},{"reference":"Wingfield JL, Lechtreck KF. 2018. Chlamydomonas Basal Bodies as Flagella Organizing Centers. Cells 7: 79.
","pubmedId":"","doi":"10.3390/cells7070079"},{"reference":"Wright RL, Salisbury J, Jarvik JW. 1985. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.. The Journal of cell biology 101: 1903-1912.
","pubmedId":"","doi":"10.1083/jcb.101.5.1903"}],"title":"Novel revertants of Chlamydomonas vfl2 suggest a role for a flexible central α-helical region in centrin function in flagellar basal bodies
","reviews":[{"reviewer":{"displayName":"Gregory J Pazour"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"c3476cba-5450-4896-9037-d1412d4abc43","decision":"accept","abstract":"A Chlamydomonas mutant, vfl2, displays aberrant flagellar numbers due to an E101K mutation in the centrin gene. We isolated 21 revertants–12 intragenic and 9 extragenic–that suppress the mutant's abnormal phenotype. AlphaFold2 modeling indicates that the wild-type, mutant, and revertant proteins are structurally similar. However, the vfl2 mutant centrin forms three salt bridges in the central region of the core α-helix, whereas the wild type and all intragenic revertants have only two salt bridges or disrupted helix continuity. These results indicate that flexibility of the central helix, maintained by limited electrostatic interactions, is important for proper centrin function.
","acknowledgements":"We thank Ms. and Mr. Takeru Ando, Miku Arai, Shota Umeki, Tomoya Oshima, and Atsuhiro Hattori (Chuo University) for their assistance with sequencing analysis. We also thank Dr. Akira Noga (Chuo University) for his critical reading of the manuscript and helpful suggestions.
","authors":[{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","fundingAcquisition","dataCuration","writing_originalDraft"],"email":"minoura.87b@g.chuo-u.ac.jp","firstName":"Takako","lastName":"Kato-Minoura","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4033-2427"},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["investigation"],"email":"i.chan.ex2017@gmail.com","firstName":"Ichiro","lastName":"Sasaki","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","supervision"],"email":"ritsu.kamiya@gmail.com","firstName":"Ritsu","lastName":"Kamiya","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Sanger sequencing data of vfl2 and 21 revertants. Only partial regions, bases 286 to 306, where the A at the start codon is 1, are shown. The red arrows indicate the vfl2-type SNP that results in the E101K mutation. The blue arrows indicate additional changes, grouped by revertant type.
","doi":null,"resourceType":"Image","name":"sup_fig_1(288).jpg","url":"https://portal.micropublication.org/uploads/c94ff44d521d0dc5efd1abdb4265d960.jpg"}],"funding":"This study was supported in part by the Chuo University Grant for Special Research.
","image":{"url":"https://portal.micropublication.org/uploads/28723abb348afeddc353c77dbb41487f.jpg"},"imageCaption":"A, Genotypes of the vfl2 revertants. B, wild-type overall centrin (Vfl2) structure (PDB: 3QRX, Sosa et al., 2011); C, magnified mid portion of wild-type centrin (box region in B); D–J, AlphaFold2-predicted centrin structures in vfl2 (D), vfl2-R201 (E), vfl2-R206 (F), vfl2-R214/R221 (G), vfl2-R220/R222 (H), vfl2-R202/R205/R217 (I), vfl2-R205 (J). In B, orange spheres indicate Ca2+ ions. Light green, dark green, light teal, and deep teal indicate the 1st to 4th EF-hand motifs, respectively. In the central α-helices in B–J, acidic/basic amino acids are shown as a stick model, with acidic amino acids (E or D) colored red and basic amino acids (K or R) colored blue. Other mutated amino acids are colored orange. In C–J, the salt bridge distances are indicated in Å, and the specific amino acid variations are shown at the bottom of each panel. The numbers in the schematic diagram of panel C indicate the amino acid positions in the wild-type primary sequence, illustrating their spatial arrangement along the pitches of the α-helix. Note that the amino acid at position 98 is not shown except in panel I, as this residue is located on the back side of the helix and not visible in the ribbon diagrams. K: alignments of the amino acid sequences of the mid portion of the central α-helix in wild type, vfl2, and type 2 and type 3 revertants.
","imageTitle":"Summary of vfl2 revertant classes and associated mutations
","methods":"Revertant isolation
The mutant vfl2 (Kuchka & Jarvik, 1982) was cultured in liquid TAP medium (Gorman & Levine, 1965) for 3 days, placed in a Petri dish, irradiated with a UV lamp (20 W) from 40 cm above for 2 min, and transferred to test tubes with 8 mL liquid TAP. After being kept in the dark for 1 day to prevent photo-recovery, the upper 0.5 mL of each test tube, containing swimming cells, was transferred to another test tube with 8 mL of fresh medium. This process was repeated a few times. Finally, the cells swimming on the upper surface of each test-tube culture were inoculated onto TAP agar plates to produce single colonies. Each clone was cultured in a multi-well plate with TAP medium for motility assessment under an inverted microscope. Strains that showed near-wild-type motility were saved as revertants.
Genomic DNA sequencing
Genomic DNA from vfl2 and its revertants was purified using the method of Rochaix et al. (1988), with some modifications. The coding region was then amplified using primers 5vfl2-F and 3vfl2-R (see Reagents/Strains). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and their sequences were determined using primers 5vfl2-F and 5vfl2-F2 (see Reagents/Strains). Sequencing was entrusted to Macrogen Japan.
3D structure prediction
AlphaFold2 (Jumper et al., 2021) was used to predict the 3D structure of vfl2 and revertants centrins, then plotted in PyMOL (DeLano Scientific,https://www.pymol.org).
","reagents":"PRIMER NAME | SEQUENCE |
5vfl2-F | 5'-AGTCTGCTGCTGGAGCTCAGACC-3 |
3vfl2-R | 5'-AATTTATTATGTGCCGTGCTGTGCAC-3' |
5vfl2-F2 | 5'-TGTGGGCATAGCGGTGACAAA-3' |
STRAIN | GENOTYPE | AVAIRABLE FROM |
CC-125 | Wild type | Chlamydomonas Resource Center |
vfl2 | E101K in Vfl2 | Chlamydomonas Resource Center (Kuchka & Jarvik, 1982; Taillon et al., 1992) |
vfl2-R201~R222 | see Figure 1A. | All revertants isolated in this study will be deposited in the Chlamydomonas Resource Center. |
Centrin is a highly conserved eukaryotic protein of ~ 20 kDa with four Ca2+-binding EF-hand motifs. In animal cells, centrin localizes to the centriole, the core structure of the centrosome, and plays essential roles in mitotic spindle formation and cytoplasmic microtubule organization. In ciliated cells, the centriole becomes a basal body and functions as the template for axonemal assembly. Centrin is thus essential for both cell division and ciliation (flagellation).
In the unicellular biflagellate green alga Chlamydomonas, centrin is expressed from the gene vfl2. It mainly localizes to three distinct sites in interphase cells: fibers connecting the nuclei and basal bodies, distal striated fibers (DSF) that connect the two basal bodies, and the transition zone located between the basal body and the flagellum (Wright et al., 1985; Schulze et al., 1987; Salisbury et al., 1988; Sanders & Salisbury, 1989). In most cases, centrin is contained in fibrous structures, which can contract at higher Ca2+ concentrations (Wingfield and Lechtreck, 2018). Centrin in the transition zone has been suggested to participate in flagellar shedding (Sanders and Salisbury, 1989), and the DSF centrin to function in changing the angle between the two flagella in a Ca2+-dependent manner (Salisbury et al., 1987; Hayashi et al., 1998).
The Chlamydomonas mutant vfl2, which displays aberrant flagellar numbers, carries a centrin-gene mutation causing a Glu-to-Lys substitution at position 101 (Kuchka & Jarvik, 1982; Taillon et al., 1992). Taillon et al. (1992) mutagenized this mutant and obtained 19 revertants that apparently recovered wild-type flagellation. Ten of these had changes at Lys101 to one of five amino acids (Taillon et al., 1992). The present study extended previous work by isolating 21 additional phenotypic revertants and identifying several novel amino acid changes (Figure 1A). All revertants displayed near-normal swimming phenotypes due to recovery of normal flagellar number. Sequence analysis revealed four classes: type 1, in which the wild-type sequence was restored; type 2, in which Lys101 was replaced by an amino acid other than the original glutamic acid; type 3, which harbored additional mutation(s) while retaining the original E101K mutation; and type 4, in which no mutation was found other than E101K. Types 1 through 3 are intragenic revertants, whereas type 4 likely reflects extragenic suppression. Some classes match those previously reported, whereas others represent newly identified revertant types.
We focus here on type 2 and type 3 mutants with intragenic changes, although type 4 mutants are also interesting and warrant future studies, as these revertants indicate mutations in non-centrin proteins. As shown in Figure 1E—J, all amino acid changes in type 2 and type 3 occurred in the central portion of the central α-helix in centrin. Within this region, electrostatic interactions occur between different amino acid side chains. For example, in the two pitches of the α-helix N-terminal to position E101, attractive interactions occur between E95 and R99, and between R96 and E100, while a repulsive interaction occurs between D97 and E101 (Figure 1B, C). In contrast, in vfl2 centrin, there are three attractively interacting amino-acid pairs: E95-R99, R96-E100, and D97-K101 (Figure 1D). This suggests that the α-helix around position 101 is more stabilized in vfl2 than in wild type, although AlphaFold2 (Jumper et al., 2021) predicts similar structures for wild-type and vfl2 centrins. All type 2 and type 3 revertants isolated in this study have either only two attractive amino-acid pairs or a proline kink in this region (Figure 1E—J). In other words, no revertants were isolated that retained the three pairs of oppositely charged amino acids present in vfl2. These results suggest that appropriate centrin flexibility—arising from limited electrostatic interaction or disruption of helix continuity—is important for centrin function at the flagellar base.
Notably, among the four types of amino acid substitutions at position 101 reported by Taillon et al. (1992)—specifically I, M, T, and N—our study isolated all but E101N. Although we did not isolate this mutation in our screen, an E101N substitution does not introduce a basic side chain and therefore cannot form the third salt bridge with D97, leaving the central helix with only two salt bridges. Thus, all revertants obtained by Taillon et al. (1992) likely have centrins with a more flexible central helix than the original vfl2 centrin, consistent with our proposed model.
Furthermore, Taillon et al. (1992) reported structural deficiencies in the DSF in some of their revertants. Although we have not examined the DSF structures in our isolated revertants, they may harbor similar defects. How central-helix flexibility specifically affects centrin function in regulating basal body number and DSF structure remains an important subject for future studies.
Based on sequence analyses of revertants, we propose that excessive stabilization of the centrin α-helix around position 101 is detrimental to its function, and that some flexibility in this region may be necessary. This idea contrasts with the proposal of Taillon et al. (1992) that E101 stabilizes the centrin structure by forming a salt bridge with another basic amino acid and that this stabilization is important for centrin function. In calmodulin (CaM), a homolog of centrin, the middle region of the corresponding central helix functions as a \"flexible tether\" crucial to its function (Persechini et al., 1988a). In CaM, the central region is almost entirely composed of negatively charged amino acids and therefore barely forms an α-helix (Persechini et al., 1988b; Barbato et al., 1992, for review, see Nelson and Chazin, 1998). Thus, some flexibility in the central region of the central helix may also be essential for centrin function. Having only two salt bridges in centrin's central helix may therefore be important for allowing this flexibility; this could be tested by generating mutants with additional salt bridges near position 101 in future studies. (Extended Data: Supplementary Figure 1)
","references":[{"reference":"Adams GM, Wright RL, Jarvik JW. 1985. Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.. The Journal of cell biology 100: 955-964.
","pubmedId":"","doi":"10.1083/jcb.100.3.955"},{"reference":"Barbato G, Ikura M, Kay LE, Pastor RW, Bax A. 1992. Backbone dynamics of calmodulin studied by nitrogen-15 relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31: 5269-5278.
","pubmedId":"","doi":"10.1021/bi00138a005"},{"reference":"Gorman DS, Levine RP. 1965. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.. Proceedings of the National Academy of Sciences 54: 1665-1669.
","pubmedId":"","doi":"10.1073/pnas.54.6.1665"},{"reference":"Hayashi M, Yagi T, Yoshimura K, Kamiya R. 1998. Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas. Cell Motil Cytoskeleton 41(1): 49-56.
","pubmedId":"9744298","doi":""},{"reference":"Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al., Hassabis. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589.
","pubmedId":"","doi":"10.1038/s41586-021-03819-2"},{"reference":"Kuchka MR, Jarvik JW. 1982. Analysis of flagellar size control using a mutant of Chlamydomonas reinhardtii with a variable number of flagella.. The Journal of cell biology 92: 170-175.
","pubmedId":"","doi":"10.1083/jcb.92.1.170"},{"reference":"Melkonian M. 1978. Structure and significance of cruciate flagellar root systems in green algae: Comparative investigations in species ofChlorosarcinopsis (Chlorosarcinales). Plant Systematics and Evolution 130: 265-292.
","pubmedId":"","doi":"10.1007/BF00982810"},{"reference":"Nelson MR, Chazin WJ. 1998. An interaction‐based analysis of calcium‐induced conformational changes in Ca2+ sensor proteins. Protein Science 7: 270-282.
","pubmedId":"","doi":"10.1002/pro.5560070206"},{"reference":"Persechini A, Kretsinger RH. 1988. The central helix of calmodulin functions as a flexible tether. J Biol Chem 263(25): 12175-8.
","pubmedId":"3137220","doi":""},{"reference":"Persechini A, Kretsinger RH. 1988. Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function. J Cardiovasc Pharmacol 12 Suppl 5: S1-12.
","pubmedId":"2469863","doi":""},{"reference":"Rochaix JD, Mayfield S, Goldschmidt CM, Erickson J. 1988. Molecular biology of Chlamydomonas. In: Shaw CH (ed) Plant molecular biology, a practical approach. IRL Press, Oxford, pp 253–275.
","pubmedId":"","doi":""},{"reference":"Salisbury JL, Baron AT, Sanders MA. 1988. The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.. The Journal of cell biology 107: 635-641.
","pubmedId":"","doi":"10.1083/jcb.107.2.635"},{"reference":"Salisbury JL, Sanders MA, Harpst L. 1987. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii.. The Journal of cell biology 105: 1799-1805.
","pubmedId":"","doi":"10.1083/jcb.105.4.1799"},{"reference":"Sanders MA, Salisbury JL. 1989. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii.. The Journal of cell biology 108: 1751-1760.
","pubmedId":"","doi":"10.1083/jcb.108.5.1751"},{"reference":"Schulze D, Robenek H, McFadden GI, Melkonian M. 1987. Immunolocalization of a Ca2+-modulated contractile protein in the flagellar apparatus of green algae: the nucleus-basal body connector. Eur. J. Cell Biol. 45: 51–61.
","pubmedId":"","doi":""},{"reference":"Sosa LdV, Alfaro E, Santiago J, Narváez D, Rosado MC, Rodríguez A, et al., Pastrana‐Ríos. 2011. The structure, molecular dynamics, and energetics of centrin–melittin complex. Proteins: Structure, Function, and Bioinformatics 79: 3132-3143.
","pubmedId":"","doi":"10.1002/prot.23142"},{"reference":"Taillon BE, Adler SA, Suhan JP, Jarvik JW. 1992. Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.. The Journal of cell biology 119: 1613-1624.
","pubmedId":"","doi":"10.1083/jcb.119.6.1613"},{"reference":"Wingfield JL, Lechtreck KF. 2018. Chlamydomonas Basal Bodies as Flagella Organizing Centers. Cells 7: 79.
","pubmedId":"","doi":"10.3390/cells7070079"},{"reference":"Wright RL, Salisbury J, Jarvik JW. 1985. A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.. The Journal of cell biology 101: 1903-1912.
","pubmedId":"","doi":"10.1083/jcb.101.5.1903"}],"title":"Novel revertants of Chlamydomonas vfl2 suggest a role for a flexible central α-helical region in centrin function in flagellar basal bodies
","reviews":[],"curatorReviews":[]},{"id":"437f8a06-58dd-4c06-a511-1faa30b227bb","decision":"publish","abstract":"A Chlamydomonas mutant, vfl2, displays aberrant flagellar numbers due to an E101K mutation in the centrin gene. We isolated 21 revertants–12 intragenic and 9 extragenic–that suppress the mutant's abnormal phenotype. AlphaFold2 modeling indicates that the wild-type, mutant, and revertant proteins are structurally similar. However, the vfl2 mutant centrin forms three salt bridges in the central region of the core α-helix, whereas the wild type and all intragenic revertants have only two salt bridges or disrupted helix continuity. These results indicate that flexibility of the central helix, maintained by limited electrostatic interactions, is important for proper centrin function.
","acknowledgements":"We thank Ms. and Mr. Takeru Ando, Miku Arai, Shota Umeki, Tomoya Oshima, and Atsuhiro Hattori (Chuo University) for their assistance with sequencing analysis. We also thank Dr. Akira Noga (Chuo University) for his critical reading of the manuscript and helpful suggestions.
","authors":[{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","fundingAcquisition","dataCuration","writing_originalDraft"],"email":"minoura.87b@g.chuo-u.ac.jp","firstName":"Takako","lastName":"Kato-Minoura","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-4033-2427"},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["investigation"],"email":"i.chan.ex2017@gmail.com","firstName":"Ichiro","lastName":"Sasaki","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo, Japan"],"departments":["Department of Biological Sciences, School of Science and Engineering"],"credit":["conceptualization","supervision"],"email":"ritsu.kamiya@gmail.com","firstName":"Ritsu","lastName":"Kamiya","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Sanger sequencing data of vfl2 and 21 revertants. Only partial regions (bases 286 to 306), where the A at the start codon is 1, are shown. The red arrows indicate the vfl2-type SNP that results in the E101K mutation. The blue arrows indicate additional changes, grouped by revertant type.
","doi":"10.22002/qpeqx-0qg84","resourceType":"Image","name":"Extended_fig_1(288).jpg","url":"https://portal.micropublication.org/uploads/0b54e27f9d4493c5081067c730b7d6ea.jpg"}],"funding":"This study was supported in part by the Chuo University Grant for Special Research.
","image":{"url":"https://portal.micropublication.org/uploads/3e2fc48585c40af6a07afbe51d58013d.jpg"},"imageCaption":"A, Genotypes of the vfl2 revertants. B, wild-type overall centrin (Vfl2) structure (PDB: 3QRX, Sosa et al., 2011); C, magnified mid portion of wild-type centrin (boxed in B); D–J, AlphaFold2-predicted centrin structures in vfl2 (D), vfl2-R201 (E), vfl2-R206 (F), vfl2-R214/R221 (G), vfl2-R220/R222 (H), vfl2-R202/R215/R217 (I), vfl2-R205 (J). In B, orange spheres indicate Ca2+ ions. Light green, dark green, light teal, and deep teal indicate the 1st–4th EF-hand motifs, respectively. In the central α-helices in B–J, acidic/basic amino acids are shown as a stick model, with acidic amino acids (E or D) colored red and basic amino acids (K or R) colored blue. Other mutated amino acids are colored orange. In C–J, salt-bridge distances are shown in Å, and the specific amino acid variations are shown at the bottom of each panel. The numbers in the schematic diagram of panel C indicate the amino acid positions in the wild-type primary sequence, illustrating their spatial arrangement along the pitches of the α-helix. Note that the amino acid at position 98 is not shown except in panel I, as this residue lies on the back side of the helix and is not visible in the ribbon diagrams. K: alignments of the amino acid sequences of the mid portion of the central α-helix in wild type, vfl2, and type 2 and type 3 revertants.
","imageTitle":"Summary of vfl2 revertant classes and associated mutations
","methods":"Revertant isolation
The mutant vfl2 (Kuchka and Jarvik, 1982) was cultured in liquid TAP medium (Gorman and Levine, 1965) for 3 days, placed in a Petri dish, irradiated with a UV lamp (20 W) from 40 cm above for 2 min, and transferred to test tubes with 8 mL liquid TAP. After being kept in the dark for 1 day to prevent photo-recovery, the upper 0.5 mL of each test tube, containing swimming cells, was transferred to another test tube with 8 mL of fresh medium. This process was repeated a few times. Finally, the cells swimming on the upper surface of each test-tube culture were inoculated onto TAP agar plates to produce single colonies. Each clone was cultured in a multi-well plate with TAP medium for motility assessment under an inverted microscope. Strains that showed near-wild-type motility were saved as revertants.
Genomic DNA sequencing
Genomic DNA from vfl2 and its revertants was purified using the method of Rochaix et al. (1988), with some modifications. The coding region was then amplified using primers 5vfl2-F and 3vfl2-R (see Reagents). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and their sequences were determined using primers 5vfl2-F and 5vfl2-F2 (see Reagents). Sequencing was entrusted to Macrogen Japan.
3D structure prediction
AlphaFold2 (Jumper et al., 2021) was used to predict the 3D structure of vfl2 and revertants centrins, then plotted in PyMOL (DeLano Scientific,https://www.pymol.org).
","reagents":"PRIMER NAME | SEQUENCE |
5vfl2-F | 5'-AGTCTGCTGCTGGAGCTCAGACC-3' |
3vfl2-R | 5'-AATTTATTATGTGCCGTGCTGTGCAC-3' |
5vfl2-F2 | 5'-TGTGGGCATAGCGGTGACAAA-3' |
STRAIN | GENOTYPE | AVAIRABLE FROM |
CC-125 | Wild type | Chlamydomonas Resource Center |
vfl2 | E101K in Vfl2 | Chlamydomonas Resource Center (Kuchka and Jarvik, 1982; Taillon et al., 1992) |
vfl2-R201~R222 | see Figure 1A | All revertants isolated in this study will be deposited in the Chlamydomonas Resource Center. |
Centrin is a highly conserved eukaryotic protein of ~ 20 kDa with four Ca2+-binding EF-hand motifs. In animal cells, centrin localizes to the centriole, the core structure of the centrosome, and plays essential roles in mitotic spindle formation and cytoplasmic microtubule organization. In ciliated cells, the centriole becomes a basal body and functions as the template for axonemal assembly. Centrin is thus essential for both cell division and ciliation (flagellation).
In the unicellular biflagellate green alga Chlamydomonas, centrin is expressed from the gene vfl2. It mainly localizes to three distinct sites in interphase cells: fibers connecting the nuclei and basal bodies, distal striated fibers (DSF) that connect the two basal bodies, and the transition zone located between the basal body and the flagellum (Wright et al., 1985; Schulze et al., 1987; Salisbury et al., 1988; Sanders and Salisbury, 1989). In most cases, centrin is contained in fibrous structures, which can contract at higher Ca2+ concentrations (Wingfield and Lechtreck, 2018). Centrin in the transition zone has been suggested to participate in flagellar shedding (Sanders and Salisbury, 1989), and the DSF centrin to function in changing the angle between the two flagella in a Ca2+-dependent manner (Salisbury et al., 1987; Hayashi et al., 1998).
The Chlamydomonas mutant vfl2, which displays aberrant flagellar numbers, carries a centrin-gene mutation causing a Glu-to-Lys substitution at position 101 (Kuchka and Jarvik, 1982; Taillon et al., 1992). Taillon et al. (1992) mutagenized this mutant and obtained 19 revertants that apparently recovered wild-type flagellation. Ten of these had changes at Lys101 to one of five amino acids (Taillon et al., 1992). The present study extended previous work by isolating 21 additional phenotypic revertants and identifying several novel amino acid changes (Figure 1A). All revertants displayed near-normal swimming phenotype due to recovery of normal flagellar number. Sequence analysis revealed four classes: type 1, in which the wild-type sequence was restored; type 2, in which Lys101 was replaced by an amino acid other than the original glutamic acid; type 3, which harbored additional mutation(s) while retaining the original E101K mutation; and type 4, in which no mutation was found other than E101K. Types 1 through 3 are intragenic revertants, whereas type 4 likely reflects extragenic suppression. Some classes match those previously reported, whereas others represent newly identified revertant types (see also Extended Data Figure 1).
We focus here on type 2 and type 3 mutants with intragenic changes, although type 4 mutants are also interesting and warrant future studies, as these revertants indicate mutations in non-centrin proteins. As shown in Figure 1E—J, all amino acid changes in type 2 and type 3 occurred in the central portion of the central α-helix in centrin. Within this region, electrostatic interactions occur between different amino acid side chains. For example, in the two pitches of the α-helix N-terminal to position E101, attractive interactions occur between E95 and R99, and between R96 and E100, while a repulsive interaction occurs between D97 and E101 (Figure 1B, C). In contrast, in vfl2 centrin, there are three attractively interacting amino-acid pairs: E95-R99, R96-E100, and D97-K101 (Figure 1D). This suggests that the α-helix around position 101 is more stabilized in vfl2 than in wild type, although AlphaFold2 (Jumper et al., 2021) predicts similar structures for wild-type and vfl2 centrins. All type 2 and type 3 revertants isolated in this study have either only two attractive amino-acid pairs or a proline kink in this region (Figure 1E—J). In other words, no revertants were isolated that retained the three pairs of oppositely charged amino acids present in vfl2. These results suggest that appropriate centrin flexibility—arising from limited electrostatic interaction or disruption of helix continuity—is important for centrin function at the flagellar base.
Notably, among the four types of amino acid substitutions at position 101 reported by Taillon et al. (1992)—specifically I, M, T, and N—our study isolated all but E101N. Although we did not isolate this mutation in our screen, an E101N substitution does not introduce a basic side chain and therefore cannot form the third salt bridge with D97, leaving the central helix with only two salt bridges. Thus, all revertants obtained by Taillon et al. (1992) likely have centrins with a more flexible central helix than the original vfl2 centrin, consistent with our proposed model.
Taillon et al. (1992) reported structural deficiencies in the DSF in some of their revertants. Although we have not examined the DSF structures in our isolated revertants, they may harbor similar defects. How central-helix flexibility specifically affects centrin function in regulating basal body number and DSF structure remains an important subject for future studies.
Based on sequence analyses of revertants, we propose that excessive stabilization of the centrin α-helix around position 101 is detrimental to its function, and that some flexibility in this region may be necessary. This idea contrasts with the proposal of Taillon et al. (1992) that E101 stabilizes the centrin structure by forming a salt bridge with another basic amino acid and that this stabilization is important for centrin function. In calmodulin (CaM), a homolog of centrin, the middle region of the corresponding central helix functions as a \"flexible tether\" crucial to its function (Persechini et al., 1988a). In CaM, the central region is almost entirely composed of negatively charged amino acids and therefore barely forms an α-helix (Persechini et al., 1988b; Barbato et al., 1992, for review, see Nelson and Chazin, 1998). Thus, some flexibility in the central region of the central helix may also be essential for centrin function. Having only two salt bridges in centrin's central helix may therefore be important for allowing this flexibility; this could be tested by generating mutants with additional salt bridges near position 101 in future studies.
","references":[{"reference":"Adams GM, Wright RL, Jarvik JW. 1985. Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number. The Journal of cell biology 100: 955-964.
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","pubmedId":"","doi":"10.1083/jcb.101.5.1903"}],"title":"Novel revertants of Chlamydomonas vfl2 suggest a role for a flexible central α-helical region in centrin function in flagellar basal bodies
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