Andréa C. Dosé

A New Compartment at Stereocilia Tips Defined by Spatial and Temporal Patterns of Myosin IIIa Expression

Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)–myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.

Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments

Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

Myosin IIIB Uses an Actin-Binding Motif in Its Espin-1 Cargo to Reach the Tips of Actin Protrusions

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

Kinetic Mechanism of Human Myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (kcat = 0.77 ± 0.08 s–1), and high actin concentrations are required to fully activate the ATPase rate (KATPase = 34 ± 11 μm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (Kactin ∼ 7 μm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K1k+2 = 0.020 and 0.001 μm–1·s–1, respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s–1) than the kcat, whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s–1). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (Kd ∼ 5 μm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.

The kinase domain alters the kinetic properties of the myosin IIIA motor.

Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.

Identification and Localization of Myosin Superfamily Members in Fish Retina and Retinal Pigmented Epithelium

Myosins are cytoskeletal motors critical for generating the forces necessary for establishing cell structure and mediating actin‐dependent cell motility. In each cell type a multitude of myosins are expressed, each myosin contributing to aspects of morphogenesis, transport, or motility occurring in that cell type. To examine the roles of myosins in individual retinal cell types, we first used polymerase chain reaction (PCR) screening to identify myosins expressed in retina and retinal pigmented epithelium (RPE), followed by immunohistochemistry to examine the cellular and subcellular localizations of seven of these expressed myosins. In the myosin PCR screen of cDNA from striped bass retina and striped bass RPE, we amplified 17 distinct myosins from eight myosin classes from retinal cDNA and 11 distinct myosins from seven myosin classes from RPE cDNA. By using antibodies specific for myosins IIA, IIB, IIIA, IIIB, VI, VIIA, and IXB, we examined the localization patterns of these myosins in retinas and RPE of fish, and in isolated inner/outer segment fragments of green sunfish photoreceptors. Each of the myosins exhibited unique expression patterns in fish retina. Individual cell types expressed multiple myosin family members, some of which colocalized within a particular cell type. Because much is known about the functions and properties of these myosins from studies in other systems, their cellular and subcellular localization patterns in the retina help us understand which roles they might play in the vertebrate retina and RPE. J. Comp. Neurol. 513:209–223, 2009.

Myosin 3A transgene expression produces abnormal actin filament bundles in transgenic Xenopus laevis rod photoreceptors.

Myo3A, a class III myosin, localizes to the distal (plus) ends of inner segment actin filament bundles that form the core of microvillus-like calycal processes encircling the base of the photoreceptor outer segment. To investigate Myo3A localization and function, we expressed green fluorescent protein-tagged bass Myo3A and related constructs in transgenic Xenopus rods using a modified opsin promoter. Tagged intact Myo3A localized to rod calycal processes, as previously reported for native bass Myo3A. Transgenic rods developed abnormally large calycal processes and subsequently degenerated. Modified Myo3A expression constructs demonstrated that calycal process localization required an active motor domain and the tail domain. Expressed tail domain alone localized to actin bundles along the entire inner segment length, rather than to the distal end. This tail domain localization required the conserved C-terminal domain (3THDII) previously shown to possess an actin-binding motif. Our findings suggest that Myo3A plays a role in the morphogenesis and maintenance of calycal processes of vertebrate photoreceptors.

Cloning and Distribution of Myosin 3B in the Mouse Retina: Differential Distribution in Cone Outer Segments

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.

Mouse class III myosins: kinase activity and phosphorylation sites.

J. Neurochem. (2011) 119, 772–784.

Chromatin and higher-order chromosome organization shape the recombination landscape in C. elegans

Meiotic recombination generates crossovers, which physically link homologous chromosomes, and direct their segregation to opposite poles. Despite the benefits, crossovers are restricted both in number as well as spatially. Crossovers in the nematode C. elegans occur most frequently within the distal regions (or “arms”) of the chromosomes, which comprise ~50% of the genome, but ~90% of the genetic map length. Meiotic recombination initiates with the formation of programmed double strand breaks (DSBs) catalyzed by the SPO-11 enzyme. It has been unclear whether the crossover bias in C. elegans is a consequence of DSB distribution, or instead reflects a mechanism that biases the downstream repair outcome of DSBs. In order to address this, we generated a genome-wide map of meiotic DSBs by mapping the distribution of the sole C. elegans RecA-like recombinase, RAD-51, by ChIP-seq. We have found that the RAD-51 binding pattern is strikingly similar to the known recombination pattern in C. elegans. The majority of RAD-51 binding sites occur on chromosome arms. Comparing RAD-51 distribution relative to functional genomic elements revealed that RAD-51 is particularly enriched in intronic DNA while coding sequences are underrepresented in RAD-51 occupied regions. We find that monomethylation of histone H3 lysine 36 (H3K36me1), which has not previously been associated with meiotic recombination, is highly correlated with break site preference. H3K36me1 is particularly enriched over active genes with high intron content, suggesting that the specific chromatin environment associated with these genes favors DSB formation in C. elegans. We have also identified a sequence motif that is highly overrepresented in DSB sites. Although the DSB pattern is highly correlated with crossover frequency, there are interesting exceptions. In particular, the sub-telomeric regions are completely devoid of crossovers but are active DSB sites. We have also identified a DNA sequence motif that appears to be enriched within DSB sites suggesting that sequence specific factors may have a role in DSB specification in C. elegans. In a parallel study, we have developed an Illumina Golden Gate SNP array to rapidly measure genome-wide recombination rates in C. elegans. Using this array we measured meiotic recombination rates in met-2(n4256) mutant animals. MET-2 is one of the two histone methyltransferases responsible for H3K9 methylation, a histone mark associated with heterochromatin. We find that the genetic map is significantly altered in met-2(n4256) mutant animals. In particular, regions that are normally enriched for H3K9 methylation show elevated recombination rates in met-2(n4256) mutants. We are currently investigating whether this change in the genetic map is the result of altered DSB distribution. Taken together, our data provide new insights into the role of chromatin in meiotic recombination. Because meiotic recombination generates genetic diversity, our data implicates chromatin as a key player in the evolution of genomes.