Tsuyoshi Sakai

The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella

The Bardet-Biedl syndrome protein complex (BBSome) is a cargo adapter rather than an essential part of the intraflagellar transport (IFT) machinery.

The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca2+ but not high Ca2+. By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca2+ suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.

Phospholipid-dependent regulation of the motor activity of myosin X

Myosin X is involved in the reorganization of the actin cytoskeleton and protrusion of filopodia. Here we studied the molecular mechanism by which bovine myosin X is regulated. The globular tail domain inhibited the motor activity of myosin X in a Ca2+-independent manner. Structural analysis revealed that myosin X is monomeric and that the band 4.1-ezrin-radixin-moesin (FERM) and pleckstrin homology (PH) domains bind to the head intramolecularly, forming an inhibited conformation. Binding of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) to the PH domain reversed the tail-induced inhibition and induced the formation of myosin X dimers. Consistently, disruption of the binding of PtdIns(3,4,5)P3 attenuated the translocation of myosin X to filopodial tips in cells. We propose the following mechanism: first, the tail inhibits the motor activity of myosin X by intramolecular head-tail interactions to form the folded conformation; second, phospholipid binding reverses the inhibition and disrupts the folded conformation, which induces dimer formation, thereby activating the mechanical and cargo transporter activity of myosin X.

Cargo binding activates myosin VIIA motor function in cells.

Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella.

The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.

Structure and Regulation of the Movement of Human Myosin VIIA

Background: Regulation of myosin VIIA (HM7A) has been studied in Drosophila but not in humans. Results: The tail domain inhibits HM7A ATPase activity and translocation to filopodial tips. Conclusion: The tail domain inhibition mechanism of HM7A is operating both in vitro and in vivo. Significance: The results provide a clue to understand the mechanism of human Usher syndrome. Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca2+. Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca2+, and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.

Phosphorylation of the kinase domain regulates autophosphorylation of myosin IIIA and its translocation in microvilli.

Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr184 and Thr188, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr184 and Thr188 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation–dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A.

Live-Cell Single-Molecule Imaging of Human Myosin IIIA

Stereocilia is an actin-based mechanosensing structure in the inner ear and plays an important role for auditory function. Myosin IIIA (MYO3A) accumulates in the tip of stereocilia and is important for structural and functional integrity of stereocilia. Loss of function of MYO3A causes progressive hearing loss. Since MYO3A is an actin-based motor protein, it has been thought that MYO3A functions as a cellular transporter of cargo molecules that create structural and functional base of stereocilia. A critical unanswered question is how MYO3A can transport its cargo molecules to constitute the structure of stereocilia. To address this question, we established live-cell single-molecule imaging of MYO3A in living cell. Deletion of kinase domain of MYO3A (MYO3AΔK) is previously shown to localize at the tip of filopodia in Hela cells. Here, we studied the dynamics of MYO3A movement at single-molecule level in cells for the first time: Kymograph analysis revealed continuous movement of MYO3AΔK towards filopodial tips. MYO3AΔKs average velocity on the filopodia in living Hela cells was ∼120 nm/s. This velocity is consistent with previous value measured by in-vitro F-actin gliding assays. In the same system single-molecule imaging of myosin X showed the velocity of ∼370 nm/s. Thus, the velocity of MYO3A was 3 times lower than that of MYO10. According to biochemical assay MYO3A is reported to have 20 times smaller rate of ATPase cycle compared to MYO10. These results suggest that in cells motor domain of MYO3A may have much faster ATPase cycle than that determined with the isolated protein.

Ca2+ Independent and Tail Dependent Regulation of the Motor Activity of Myosin X

Myosin X is involved in the actin cytoskeletal reorganization and protrusion of filopodia. Here we studied the molecular mechanism of regulation of myosin X. The actin-activated ATPase activity of M10Full was Ca2+ independent and significantly lower than that of M10HMM. The tail domain significantly inhibited the actin-activated ATPase activity of M10HMM regardless of Ca2+. The inhibition showed significant dependence on salt concentration, suggesting that the inhibition is dependent on ionic interaction between the tail domain and the head/neck domain of myosin X. The in vitro actin gliding velocity was markedly inhibited (4 fold) by the tail. These results suggest that the tail domain functions as an intra-molecular inhibitor of the myosin X motor function. The deletion of FERM domain abolished the inhibitory activity of the tail. On the other hand, deletion of the N-terminal PEST domain did not affect the inhibitory activity. Further truncation of the PH domain abolished the inhibitory activity of the tail. These results suggest that both the PH and FERM domains of the tail are required for the inhibition. On the other hand, the elimination of both IQ domains and the SAH/coiled-coil domain showed no effect on the tail induced inhibition. Furthermore, M10IQo co-immunoprecipitated with M10PH-FERM. The result indicated that the tail domain (PH-FERM) directly interacts with the motor domain to inhibit the motor activity. Electron microscopy revealed that the full-length myosin X molecules were monomeric, showing the wider molecules in low salt with ATP, while narrow molecules, similar head shapes to the M10HMM, in high salt. Our observation suggested that the tail domain folds backward to the head, such that it appeared to interact with the motor domain, and thus inhibits the motor activity of myosin X.(Supported by NIH).