Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

Abstract

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9+2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.

Figure 1. Cross section of ciliary and axonemal structure

A cross-section through a Chlamydomonas cilium shows the ciliary membrane which surrounds the internal axoneme structure. The axoneme is composed of a specific “9 + 2” arrangement of microtubules; 9 outer doublet microtubules, each comprised of an A- and B-tubule, surrounding a pair of internal microtubules called the central pair (CP; C1 and C2). The central pair has associated structures (grey), which transiently contact the radial spokes (RS) to generate signals which regulate the inner and outer dynein arm motors (ODA and IDA). Connecting the outer doublets are structures including the DRC-nexin link and the 5–6 bridge. The line through the cilium depicts a structural and functional ciliary axis. Dyneins on one side of the axis (on the orange outer doublets) are active for bending in one direction. When the direction of bending changes, the location of dynein activity switches; i.e. the active dyneins (on the orange outer doublets) become inactive and the dyneins on the opposite side of the axis (on the blue outer doublets) become active.

Figure 2. Predicted location of axonemal signaling proteins

The diagram depicts a cross-section view of the central pair, one radial spoke and one outer doublet microtubule. The outer and inner dynein arms (ODA and IDA respectively) are anchored to the outer doublet microtubules. Regulatory kinases and phosphatases include PP1 (located primarily in the central pair with a small amount on the outer doublets, possibly near the ODA), PP2A (located on the outer doublets, possibly near I1 dynein), CK1 (located on the outer doublets, possibly near I1 dynein), and PKA (predictably located at the base of the radial spoke and in the central pair by the RSP3-AKAP and AKAP240 respectively). Also shown are the predicted locations of calcium-sensitive regulatory complexes that contain calmodulin.

Figure 3. Schematic of a longitudinal view of the 96-nm repeat

A longitudinal view of an outer doublet microtubule with the dynein motors (ODA and IDA), radial spokes (S1 and S2), and the DRC-nexin link. These structures are arranged in a precise manner which forms a 96-nm structural element that repeats along the entire length of the axoneme. Within each 96-nm repeat, there are four ODA, two RS, one DRC-nexin link (DRC), and one copy each of several different types of IDA motors. The IDA shown in red is called I1 dynein (also known as dynein-f). This particular motor is regulated by a phosphorylation by a mechano-chemical mechanism that involves the central pair and radial spoke structures.

Figure 4. Dynein-driven microtubule sliding in cilia and flagella

The dynein arms attach to the A-tubules of the outer doublets and the motor heads transiently interact with the B-tubules of the adjacent outer doublets. Movement of the motor domains in the minus end direction toward the base of the cilium (white arrows arrows on the red dynein heads) causes the A-tubule of one doublet to slide toward the base of the adjacent B-tubule pushing that outer doublet tipward (black arrow). Because both outer doublets are anchored at the minus end by the basal body and connected along the length by the DRC-nexin links, this sliding movement forces the outer doublets to bend, resulting in a ciliary bend.

Figure 5. Model for regulation of I1 dynein by phosphorylation

I1 dynein activity is regulated by phosphorylation of IC138. When phosphorylated, I1 dynein is inactive; upon dephosphorylation, I1 dynein is active. The kinases and phosphatases involved were identified based on pharmacological data using inhibitors and include CK1, PKA, PP1 and PP2A. The central pair (CP) and radial spokes (RS) function, in part, to regulate the kinases in vivo; when mutated, the kinases are constitutively active, resulting in uniform phosphorylation of I1 dynein and global inactivation of dynein activity. This inactivation can be relieved, in vitro, by the addition of kinase inhibitors. Consequently, in vitro inhibition of phosphatase activity prevents dynein activation.