A Mutation in the RII-binding Domain of RSP3 Results in Decreased Flagellar Motility
To determine the physiological role of RII binding by RSP3, the RSP3 gene was mutated in the region coding for the RII-binding domain (). The RII-binding domain was altered as described previously for the in vitro studies of RSP3, in which valine 169 and leucine 170 were replaced by alanines. Both the mutated RSP3 gene and the wt RSP3 gene (as a control) were transformed into pf14
cells that lack axonemal RSP3 and radial spokes. Several independent transformants were obtained for each gene, and the axonemes from the transformants were analyzed for the presence of RSP3 and radial spokes. Western blot analysis of the axonemes from the transformants revealed that both the wt transformants (323, 337, 357, and 434) and the mutant transformants (44, 214, and 388) contain axonemal RSP3 (A, top). Axonemal RSP3 of the mutant transformants migrates slightly slower on an SDS-PAGE gel compared with axonemal RSP3 of the wt transformants, consistent with the in vitro studies of RSP3 described previously (Gaillard et al., 2001
), thereby providing a marker for the mutant protein. RII overlay analysis of axonemal protein from the transformants revealed that wt axonemal RSP3 binds to RII, whereas, as predicted, the mutant form of axonemal RSP3 fails to bind to RII; again, consistent with previous in vitro studies of RSP3 (A, bottom).
Figure 2. Analysis of pf14/nit1-305 cells transformed with the RSP3 gene: Pf14/nit1-305 cells were transformed with either a wt or mutant (V169L170→AA) RSP3 gene, and independent transformants were isolated and characterized. (A) Western blot and RII overlay (more ...)
Transformants were then randomly selected for further study, with particular focus on analysis of motility. To determine the degree of motility for each cell type, individual cells were scored as motile or immotile, and a percentage of motility was obtained for each cell type. Using this assay, ~90% of wt cells were motile (B). Similarly, ~90% of the wt RSP3 transformants (357 and 434) were motile, confirming successful transformation rescue of pf14 (B). In striking contrast, the mutant RSP3 transformants (214 and 388) displayed an unusual mixed motility phenotype: ~50% of the cells were motile and ~50% were immotile (B). Besides a quantitative difference in motility between the wt and mutant transformants, qualitative differences in motility were also discerned. Observations of the mutant RSP3 transformants revealed that the motile cells are comprised of normally swimming cells as well as spinning or regularly twitching cells. The spinning cells were observed to be biflagellate and presumably are the result of cells having one motile flagellum and one immotile flagellum. Measurements of swimming speed and beat frequency were performed for the normally swimming mutant RSP3 transformants, revealing the same swimming speed and flagellar beat frequency as wild-type cells.
To ensure that the mutant RSP3 transformant cells are genetically identical and not a mixture of different genotype populations, the cells were subcloned several times. To do this, a single mutant RSP3 transformant cell was isolated and then allowed to multiply into a population of cells. The population of cells was then scored for motility, and in all cases the population of cells exhibited a mixed motility phenotype (nonmotile, spinning, normal swimming). Additional experiments showed that the mixed motility phenotype of the mutant RSP3 transformants is unrelated to cell culture density or temperature and is also present when the cells are gametes. Collectively, these experiments demonstrate that the mixed motility phenotype is an inherent characteristic of the mutant RSP3 transformant cells. For convenience, and because the mutant cells display ~50% motile and 50% immotile cells, we refer to the mutant phenotype as the “50:50 phenotype.”
To make certain that the decreased motility of the mutant RSP3 transformants is due to the presence of the mutant RSP3 gene, 388 cells were backcrossed to wild-type cells, and tetrad analysis was performed. Each tetrad progeny was scored for motility and for the presence of RSP3 in the axoneme (assessed by Western blots). Because the mutated version of the RSP3 gene produces axonemal RSP3 protein that migrates more slowly on an SDS-PAGE gel, the presence of the mutant RSP3 gene can be easily distinguished from that of the wild-type RSP3 gene. Tetrad analysis revealed approximately equal numbers of parental ditype tetrads (PD) and nonparental ditype tetrads (NPD). Thus, the transformed mutant RSP3 gene is most likely located on a separate chromosome from the RSP3 gene found in wt cells. The motility of each of the tetrad progeny was then measured and recorded as a percentage of motility. In all cases, the 50:50 motility phenotype exclusively cosegregated with the mutant RSP3 gene, demonstrating a definitive linkage between the mutant RSP3 gene and the 50:50 motility phenotype. For example, progeny 2A and 2D, of a randomly selected PD tetrad, exhibit both decreased motility and the presence of the mutant RSP3 gene, whereas progeny 2B and 2C contain the wt RSP3 gene and have motility similar to wt cells (, A and B).
Figure 3. Genetic analysis of a mutant RSP3 transformant: 388 cells were mated with wt cells and tetrad analysis was performed, revealing a relatively equal occurrence of parental ditype (PD) and nonparental ditype (NPD) tetrads. (A) Western blot analysis of axonemes (more ...)
The Mutation in the PKA-binding Domain Does Not Affect Radial Spoke Assembly
To determine whether the defective motility of the mutant RSP3 transformants is due to a defect in radial spoke assembly, axonemes from mutant RSP3 transformants were isolated and compared with isolated axonemes from wt RSP3 transformants, wt cells, and pf14 cells. Electron microscopy was performed on the axonemes, and electron microscopy images of both cross and longitudinal sections revealed that radial spokes are present in both wt (357) and mutant (388) RSP3 transformants, resembling the appearance of radial spokes in wt cells (A). Radial spokes are clearly not present in pf14 cells, showing that transformation of the cells with the RSP3 gene (wt or mutant) is responsible for restoring radial spoke assembly (A). Because a lack of radial spokes in the axoneme results in displacement of the central pair apparatus, the position of the central pair can be used as an assay for the presence of assembled radial spokes (see A for reference). Axonemal cross-sections from wt cells, pf14 cells, wt RSP3 transformant cells (357 and 434), and mutant RSP3 transformant cells (214 and 388) were randomly selected, and the position of the central pair was recorded as central or displaced. The assay demonstrated that wt cells, wt RSP3 transformants (357 and 434), and mutant RSP3 transformants (214 and 388) all have axonemes that predominantly contain centered central pair apparatuses (B), consistent with the presence of radial spokes in the axoneme. In contrast, pf14 cells have axonemes that contain mostly displaced central pair apparatuses (B), a characteristic feature of axonemes lacking radial spokes. Axonemes were also analyzed by 2-D gel electrophoresis, focusing on radial spoke proteins 1-7, and analysis revealed that both wt (357) and mutant (388) RSP3 transformants contain wild-type levels of radial spoke proteins (C). In contrast, pf14 axonemes lack radial spoke proteins (C). Together, these three independent measures for the presence of radial spokes all demonstrate that the mutant RSP3 transformants contain normal amounts of radial spokes; thus, radial spoke assembly is not affected by the mutation in the PKA-binding domain of RSP3.
Figure 4. Structural analysis of axonemes from wt and mutant transformants: (A) Electron microscopy of axonemes from randomly selected wt and mutant RSP3 transformants. Cross-sections (left) and longitudinal sections (right) are shown. Both wt (357) and mutant (more ...)
Once the Flagellum Assembles, Individual Cells Do Not Switch between Motile and Immotile States
To further study the “50:50” motility phenotype and test whether individual cells can switch between motile and immotile states, mutant transformant cells were fractionated in small cultures to enrich for motile and immotile populations. The percentage of motility for the motile and immotile fractions of cells was then measured over time. The fractionation procedure was partially successful: before fractionation the cells were ~50% motile (, bar 1), whereas after fractionation, the motile fraction displayed ~80–90% motility (, bar 2) and the immotile fraction displayed ~25–30% motility (, bar 6). After fractionation, the cells were incubated for up to 8 h and periodically observed, and motility was measured again. The overall degree of motility for the motile and immotile fractions of cells remained unchanged over time (our unpublished data). This observation shows that individual cells do not switch between motile and immotile states once the flagellum is assembled, indicating that at the time of assembly a flagellum is either motile or immotile. Consistent with this idea, the ratio of motile cells that display motility of both flagella to those that display motility in just one flagellum (as revealed by a spinning motion) was also observed to remain constant over time.
Figure 5. Deflagellation and flagellar regeneration of motile and immotile fractions of a mutant RSP3 transformant: 388 cells were assessed for motility (live, bar 1) and then separated into two fractions: motile (388M) and immotile (388IM). The fractionated cells (more ...)
To further test the idea that motility is determined at the time of flagellar assembly, we used a flagellar regeneration strategy. We predicted that deflagellation of the enriched motile cells (showing ~90% motility) would restore the cells to 50% motility, and that, similarly, the “immotile” fraction (showing ~25% motility) would be restored to 50% motility. To test this prediction, each fraction of cells was deflagellated by pH shock, and the motility of the cells was observed at 0, 3, and 6 h after deflagellation. Immediately after deflagellation, both fractions of cells were observed to be immotile, due to the absence of flagella (, 0 h, bars 3 and 7). At 3 h after deflagellation, the motile fraction of cells exhibited ~40% motility, whereas the immotile fraction exhibited ~25% motility (, 3 h, bars 4 and 8). As predicted, 6 h after deflagellation, the motile fraction of cells showed ~60% motility and the immotile fraction ~55% motility (, 6 h, bars 5 and 9). Thus, after deflagellation and then regeneration of the flagella, the 50:50 motility phenotype was restored.
PKA Inhibitors Rescue Motility of Mutant RSP3 Transformant Cells
The mutation in RSP3 was designed to interrupt PKA binding (). Therefore, we postulated that the mixed motility phenotype of the transformants, particularly the immotility (Hasegawa et al., 1987
), is a consequence of misregulated axonemal PKA. To test this, and to further assess whether motility is a stable feature of each axoneme, in vitro-reactivated cell motility experiments were conducted. The motility of wt cells, wt RSP3 transformants (357), and mutant RSP3 transformants (388) was observed, and, as expected, motilities of ~95, 95, and 55% were revealed, respectively (A, live, bars 1, 4, and 7, respectively). Cells were then demembranated and motility was reactivated in a buffer containing 1 mM ATP. For all three cell types, the degree of motility for the reactivated cells was strikingly similar to that of the live cells (A, compare live versus react). In particular, both the live and reactivated mutant transformants (A, 388, bars 7 and 8) displayed the same 50:50 mixed motility phenotype for live and reactivated cells. This result further indicates that the difference between motile and immotile fractions of cells is established when the axoneme is assembled and is a stable feature of axonemal structure.
Figure 6. Pharmacological analysis of RSP3 transformants using demembranated cell models. (A) PKI-induced rescue of motility for mutant RSP3 transformant cells. Live cell motility of wt cells, wt transformant (357) cells and mutant transformant (388) cells (live) (more ...)
The result of the reactivation experiments also permitted us to further test the idea that immotility is due to misregulation of axonemal PKA in the mutant transformants. This hypothesis is based on the design of the experiment—disruption of the PKA-binding domain of RSP3 in the mutant transformants—and the fact that increased PKA activity is known to inhibit dynein activity and axonemal motility (Hasegawa et al., 1987
; Howard et al., 1994
; Habermacher and Sale, 1996
; Smith, 2002
). Therefore, we predicted that addition of PKA inhibitors, including PKI and exogenous RII, would rescue motility in the immotile fraction of reactivated cells. To test this prediction, cell models were generated and reactivation of cell motility was performed in the presence or absence of the PKA peptide inhibitor PKI (Howard et al., 1994
). Strikingly, the motility of the mutant RSP3 transformant cells increased from ~55 to ~85% in the presence of 50 nM PKI (A, +
PKI, bar 9). In contrast, the motilities of the wt cells and wt RSP3 transformant cells remained relatively unchanged (A, + PKI, bars 3 and 6, respectively). The motility of reactivated mutant RSP3 transformants was also increased significantly to ~75% upon the addition of 50 nM RII (A, + RII, bar 10; Howard et al., 1994
). As a control of specificity, the RII-induced rescue of motility was suppressed by the addition of 5 μM cAMP (A, +
RII/cAMP, bar 11). Moreover, and as expected, cAMP alone had no effect on motility of 388 mutant transformant cells. Together, these experiments suggest that the motility defect exhibited by the mutant RSP3 transformant cells is caused by unregulated and inappropriately active flagellar PKA.
Rescue of Motility with PKA Inhibitors Also Requires the Activity of a Flagellar Phosphatase
On the basis of previous studies, including data showing that PKA activity is inhibitory for the motility of Chlamydomonas
flagella, we postulated that rescue of motility for the mutant RSP3 transformants would also require the activity of a flagellar phosphatase (Hasegawa et al., 1987
; Habermacher and Sale, 1996
; Yang et al., 2000
). To test this, additional experiments using reactivated cell models were performed in the presence of the phosphatase inhibitor MC (Habermacher and Sale, 1996
; Yang et al., 2000
). We predicted that the addition of MC would block PKI-induced rescue of motility for the mutant RSP3 transformant cells. Reactivation of wt cells and mutant RSP3 transformant cells was performed, and, as expected, the reactivated cells had motilities similar to that of the live cells. About 90% of live and reactivated wt cells were motile and ~50% of the live and reactivated mutant RSP3 transformant cells were motile (B, live, bars 1 and 4, respectively). As before, addition of 50 nM PKI partially rescued reactivated motility of the mutant RSP3 transformants (B, + PKI, bar 6). However, when cell motility was reactivated in the presence of both 50 nM PKI and 1 μM MC, rescue of motility was blocked (B, compare bar 6 with bars 8 and 9). As a control, reactivation was performed in the presence of 1 μM MC only. For both wt cells and mutant RSP3 transformant cells, MC alone had little effect on motility (B, bars 3 and 7). Therefore, MC blocked the motility-rescuing effects of PKI, indicating that a flagellar phosphatase is required for rescue of motility.