Browsing by Subject "Myosin"

Mutations in myosin VIIa (MyoVIIa), an unconventional myosin, have been shown to cause Usher Syndrome Type 1B in humans. Usher Syndrome Type 1B is characterized by congenital sensorineural deafness, vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. Mouse model studies show that sensorineural deafness and vestibular dysfunction in MyoVIIa mutants is caused by disruption in the structure of microvilli-like projections (stereocilia) of hair cells in the cochlea and vestibular organ. MyoVIIa has also been shown to affect adaptation of mechanoelectrical transduction channels in stereocilia.

In Drosophila melanogaster mutations in MyoVIIa encoded by crinkled (ck) cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in Drosophila melanogaster to investigate the molecular basis of ck/MyoVIIa function and its regulation. We use live time-lapse confocal microscopy and genetic manipulations to investigate the requirement of ck/MyoVIIa function in various steps of morphogenesis of hairs and bristles. Here we show that null or near null mutations in ck/MyoVIIa lead to the formation of 8-10 short and thin hairs (split hairs) per epithelial cell that are likely the result of the failure of association of hair-actin bundles that in wild-type cells come together to form a single hair.

The myosin super family of motor proteins is divided into 17 classes by virtue of differences in the sequence of their motor domain, which presumably affect their physiological functions. In addition, substantial variety in the overall structure of their tail plays an important role in the differential regulation of myosin function. In this study we show that ck/MyoVIIa, that has two MyTH4 FERM domains in its tail separated by an SH3 domain, requires both MyTH4 FERM repeats for efficient association of hair-actin bundles to form hairs. We also show that the "multiple hair" phenotype of over-expression of ck/MyoVIIa requires both MyTH4 FERM domain function but not the tail-SH3 domain. We further demonstrate that the tail-SH3 domain of ck/MyoVIIa plays a role in keeping actin bundles, which run parallel to the length of the growing bristle, separate from each other. Our data also suggests that the tail-SH3 domain plays a role in the association of the actin filament bundles with the membrane and regulates F-actin levels in bristles.

We further demonstrate that over-expression of Quail (villin) can rescue the hair elongation defects seen in ck/MyoVIIa null or near null mutants but does not rescue the split hair defects. We show that over-expression of Alpha-actinin-GFP, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of Alpha-actinin-GFP in a ck/MyoVIIa null or near null background shows that Alpha-actinin-GFP cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing Alpha-actinin-GFP in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that Alpha-actinin-GFP over-expression causes the formation of more than the normal complement of hair-actin bundles per cell, resulting in a multiple hair phenotype. We show that Twinfilin, an actin monomer sequestering protein implicated in negatively regulating F-actin bundle elongation in stereocilia in a MyoVIIa-dependent manner, is required for F-actin bundle stability.

In addition, we use yeast two-hybrid strategies to identify Slam as a protein that directly binds to ck/MyoVIIa. We show that Slam, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of Slam and loss-of-function mutations in Slam phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of Slam and RhoGEF2 association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via RhoGEF2.

Together our results show that ck/MyoVIIa plays an important role in regulating the actin cytoskeleton that underlies actin-based cellular protrusions like hairs and bristles.

Over the last decade, a growing number of fungal infections of animals and plants has risen and persisted. The lack of diversity in antifungal drugs as well as rising antifungal resistance in many pathogens has exacerbated the problem. Thus there is a critical need for basic molecular understanding of fungal morphogenesis and pathogenesis to design new ways to combat these diseases.

One of the fungal infections in need of new treatments is Aspergillus fumigatus, the etiological agent of invasive aspergillosis. A. fumigatus is an obligate filamentous fungus that is commonly found in the soil and air. It generally does not cause invasive disease in immunocompetent hosts; however immunocompromised people are at risk for invasive aspergillosis. To better understand the morphogenesis and pathogenesis of this fungus, I decided to study myosins, a group of actin-based motor proteins that are involved in myriad of integral processes in other organisms.

Through gene deletion, I revealed the importance of the class II myosin, MyoB, in septal formation and conidiation. The class V myosin, MyoE, was required for hyphal polarity, radial extension, septal frequency and conidiation. Importantly, MyoE was required for full virulence in a murine model of invasive aspergillosis. Given the importance of MyoE in critical processes such as hyphal growth and pathogenesis, I aimed to understand the molecular requirements of MyoE. Through iterative truncations of MyoE’s N-terminal tail domain, I revealed the importance of the tail domain in hyphal growth, polarity, and MyoE localization. I identified several phosphorylated residues on the MyoE protein, but mutational analysis did not reveal that any one residue was required for MyoE function. In the absence of the serine/threonine phosphatase, calcineurin, MyoE was phosphorylated at an additional residue. Mutational analysis of a residue in the tail domain revealed it was required for septal localization but not hyphal tip localization, growth, or septation.

Because MyoE is a cargo binding protein, it likely participates in several pathways that are required for growth and septation of the fungus. To identify novel roles of class V myosins, I identified the MyoE interactome using LC-MS/MS analysis. This analysis revealed several components of the COPII pathway for ER to Golgi transport, suggesting that MyoE may play a role in this protein transport system. My future work aims to understand this role.

Epithelial sheet morphogenesis is characterized by dynamic tissue movements, resulting in the recognition and adhesion of cells to generate a seamless epithelium. Each step is mediated by carefully organized, cellular actin structures, including contractile purse strings, cellular protrusions, and dynamic medioapical arrays. I used live, 4D imaging to observe Drosophila dorsal closure, a model of epithelial sheet morphogenesis. I compared four fluorescently tagged F-actin probes widely used by Drosophila researchers to determine which was optimal for imaging dorsal closure. I observed differences in the intensity of the probes and the viability of the stocks that carry them. I quantified the rate of closure and the oscillatory behavior of amnioserosa cells when embryos expressed each F-actin probe. My findings demonstrated that each probe can be used to image F-actin during dorsal closure, and that the effects of probe expression make one probe more or less suitable than another for answering specific questions. I investigated the structure, kinematics and location of medioapical, actomyosin arrays during dorsal closure. I resolved medioapical arrays in vivo at the level of individual cytoskeletal components using total internal reflection structured illumination microscopy (TIRF-SIM). In concert with lattice light-sheet images, I show that when amnioserosa cells are relaxed, actin and myosin form a loose, domed meshwork that protrudes apically from the cellular junctions to which they are anchored. As the amnioserosa cells contract, this meshwork condenses, rearranges and is drawn basally towards the plane of the junctional belts. As the cells relax, so too does the actin and myosin meshwork in a new configuration. The medioapical arrays are juxtaposed to the plasma membrane and continuous with the extending lamellipodia and filopodia. Thus, medioapical arrays are modified cell cortex.