IC138 defines a subdomain at the base of the I1 dynein that regulates microtubule sliding and flagellar motility

Abstract

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120--defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

Figure 1.

Proposed organizational structure of I1 dynein within 96-nm axoneme repeat. The schematic diagram shows the proposed location of the IC/LC complex based on previous studies of I1 dynein motor domain mutants (Myster et al., 1998; Perrone et al., 2000; modified from Porter and Sale, 2000).

Figure 2.

Deletion of the IC138 gene in the bop5-2 strain. Shown here is a schematic diagram of the intron-exon structure of the IC138 gene in wild-type and the deleted region in bop5-2. PCR with gene-specific primers demonstrated that only the 5′ end and first exon of the IC138 gene is retained in bop5-2. Additional PCR reactions and Southern blots showed that the deletion extends ∼15 kb beyond the 3′ end of the IC138 gene.

Figure 3.

Identification of an IC138 subcomplex within the I1 dynein. (A) Schematic diagrams of the IC138 subcomplex in I1 dyneins from wild-type, bop5-1, bop5-2, and IC138 rescued (bop5-2::IC138) strains. The other I1 dynein LCs are not shown here. (B) Western blot of isolated axonemes from wild-type and mutant cells. Note the presence of IC140 and the 1α HC in the bop5-2 axonemes, but the absence of IC138. As reported previously, IC138 is truncated in bop5-1 and shifted by hyperphosphorylation in mia1 and mia2. The outer arm subunit IC69 serves as a loading control. (C) Western blot of isolated axonemes from wild-type, bop5-2, and IC138 rescued cells. IC138, IC97 and FAP120 are missing in bop5-2 axonemes, but all three polypeptides are restored in axonemes from the IC138 rescued strain. Note that the MBO2 protein is not part of the IC138 subcomplex, because it missing in axonemes from both bop5-2 and the IC138 rescued strain.

Figure 4.

Dissociation of the I1 dynein complex in bop5-2 dynein extracts. (A) Western blot of sucrose gradient fractions from a wild-type dynein extract probed with antibodies against several I1 dynein subunits. The I1 dynein subunits cosediment and peak at >20 S. FAP120 does not copurify with I1 dynein after salt extraction of wild-type axonemes (Ikeda et al., 2009) and thus was not analyzed here. (B) Western blot of sucrose gradient fractions from a bop5-2 dynein extract probed with antibodies against I1 dynein subunits. IC140 and the 1α DHC cosediment at >12 S, but the I1 dynein LCs Tctex1 and Tctex2b dissociate and sediment near the top of the sucrose gradient.

Figure 5.

Defects in I1 structure in bop5-2 axonemes. Top row, averages of the 96-nm axoneme repeat from wild-type, bop5-2, and IC138 rescued (bop5-2::IC138) cells, based on six, six, and nine individual axonemes and 61, 63, and 93 repeating units, respectively. The proximal end of the repeat is on the left, the outer arms (OA) are shown on the top, and the two radial spokes (S1 and S2) are shown on the bottom. The I1 dynein is the trilobed structure at the proximal end of the repeat. The density of the third lobe near the base of S1 is reduced in bop5-2 and restored in the IC138 rescued (bop5::IC138) strain. Bottom row, diagram of densities within the 96-nm repeat and difference plots showing a statistically significant difference in the third lobe of the I1 dynein in bop5-2 (see arrow).

Figure 6.

IC138 is required for regulated microtubule sliding. (A) Microtubule sliding disintegration assays indicate that bop5-2 axonemes display slow microtubule sliding velocities relative to wild type. Sliding velocities increase to wild-type levels in the IC138 rescued strains (bop5-2::IC138). (B) Pretreatment of radial spoke mutants with kinase inhibitors increases microtubule sliding velocities only in presence of the IC138 subcomplex (pf17 and pf17 bop5-2::IC138). Kinase inhibitors have no effect in the absence of the IC138 subcomplex (pf17 bop5-2). Microtubule sliding velocities are expressed as micrometers per second. The average microtubule sliding velocity was calculated from three independent experiments, each with a sample size of at least 70 axonemes. Values shown are means and standard deviations.

Figure 7.

Model of the IC138 subcomplex and its role in regulating I1 dynein activity. (A) Shown here is a schematic diagram of the I1-dynein subunits on the A-tubule of the outer doublet. IC138 forms a regulatory subcomplex with LC7b, FAP120, and IC97. Attachment of the IC138 subcomplex to the outer doublet may be facilitated by IC97, which also interacts with tubulin and LC8 (Wirschell et al., 2009). Attachment of the remaining I1 subunits to the A-tubule may be facilitated by IC140 (Perrone et al., 1998; Yang et al., 1998). The precise locations of the other LC subunits are unknown. (B) Diagram showing the role of the IC138 subcomplex in regulating I1 dynein activity in response to signals from the radial spoke complex. In wild-type and IC138 rescued cells, the I1 dynein is active and microtubule sliding is fast. Radial spoke mutations result in hyperphosphorylation of IC138, inhibition of I1 activity, and reduced microtubule sliding velocities. Pretreatment of axonemes with kinase inhibitors results in dephosphorylation of IC138 by endogenous phosphatases, stimulation of I1 activity, and increased microtubule sliding. In the absence of the IC138 subcomplex, the I1 dynein is inactive under all conditions.