Molecular regulatory mechanism of human myosin-7a.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

Applying Generalizability Theory to Optimize Analysis of Spontaneous Teacher Talk in Elementary Classrooms.

Purpose Although sampling teachers' child-directed speech in school settings is needed to understand the influence of linguistic input on child outcomes, empirical guidance for measurement procedures needed to obtain representative samples is lacking. To optimize resources needed to transcribe, code, and analyze classroom samples, this exploratory study assessed the minimum number and duration of samples needed for a reliable analysis of conventional and researcher-developed measures of teacher talk in elementary classrooms. Method This study applied fully crossed, Person (teacher) × Session (samples obtained on 3 separate occasions) generalizability studies to analyze an extant data set of three 10-min language samples provided by 28 general and special education teachers recorded during large-group instruction across the school year. Subsequently, a series of decision studies estimated of the number and duration of sessions needed to obtain the criterion g coefficient (g > .70). Results The most stable variables were total number of words and mazes, requiring only a single 10-min sample, two 6-min samples, or three 3-min samples to reach criterion. No measured variables related to content or complexity were adequately stable regardless of number and duration of samples. Conclusions Generalizability studies confirmed that a large proportion of variance was attributable to individuals rather than the sampling occasion when analyzing the amount and fluency of spontaneous teacher talk. In general, conventionally reported outcomes were more stable than researcher-developed codes, which suggests some categories of teacher talk are more context dependent than others and thus require more intensive data collection to measure reliably.