Centrin is a highly conserved eukaryotic protein of ~ 20 kDa with four Ca ²⁺ -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 Ca ²⁺ 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 Ca ²⁺ -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.

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).