A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells.

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a Gα12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation.

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here we identify a myosin light-chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endoderm precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, ß-catenin, and cadherin become highly enriched at the apical junctions of apically constricting cells and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis.

Gα12 structural determinants of Hsp90 interaction are necessary for serum response element-mediated transcriptional activation.

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify "class-distinctive" residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)-mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA-mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog.

Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells and used RNA interference-based screening to identify Mist, a Drosophila G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes.

Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Heterotrimeric G proteins, composed of α, ß, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

Including service learning in the undergraduate communication sciences and disorders curriculum: benefits, challenges, and strategies for success.

PURPOSE: To describe some of the benefits of service learning (SL), considerations in course development and construction, and implementation and outcomes of an SL course in the undergraduate communication sciences and disorders (CSD) program at a small, public university in northwest Washington. METHOD: A review of the literature on SL and a description of the author's experience in course development are provided on the basis of a computerized database search, library search, and discussions with the Western Washington University Center for Service Learning. CONCLUSIONS: Teaching an SL course can present challenges to both faculty and students; nonetheless, incorporating SL into the undergraduate CSD curriculum is an excellent way of enriching the academic experience and improving critical-thinking skills of young students. SL provides hands-on opportunities for students to apply what they are learning in their CSD classes to real-world contexts, gain a better understanding of course content through engagement in real situations, and integrate information from a variety of courses in and outside of their major.

Acoustic and perceptual appraisal of speech production in pediatric cochlear implant users.

OBJECTIVE: The purpose of the present study was to examine the relationship between objectively measurable acoustic changes in speech production and subjective speech production accuracy and perceived intelligibility immediately following a disruption in auditory feedback normally provided to subjects from a cochlear implant. METHODS: Six children with profound sensorineural hearing loss participated in the study. Their task was to produce speech samples in two conditions: (1) with auditory feedback from their cochlear implants, and (2) without auditory feedback from their cochlear implants. Samples were subjected to both objective and subjective analyses. Objectively, measures were made of duration, fundamental frequency, and the first and second formants of the vowels. Subjectively, two groups of listeners, one familiar with the speech of children with hearing loss and the other unfamiliar, transcribed the productions and provided ratings of intelligibility. RESULTS: All the children in this study exhibited significant differences from the cochlear implant-on to the cochlear implant-off condition, although these changes were not always in the predicted direction, nor were they always perceptually salient. CONCLUSIONS: Consistent with previous studies, children in this investigation demonstrated variable acoustic voice and speech changes following deactivation of their cochlear implant device. Few of these acoustic changes affected speech intelligibility. The results of this study overall suggest that during the initial years following implantation children who are deaf rely to some extent on the auditory feedback provided by a cochlear implant to control and modify F0, duration, and vowel formant production.