Browsing by Author "Kiehart, Daniel P"

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.

Dorsal closure in Drosophila embryos provides an excellent model system for the analysis of the coordinated cell shape changes and biomechanical processes that drive morphogenesis. During closure, the dorsal surface of the embryo displays an eye–shaped structure consisting of amnioserosa flanked by sheets of lateral epidermis. The canthi are found at the corners of the eye–shaped dorsal opening and are the focus of this dissertation. A synthesis of the four biomechanical processes that contribute to dorsal closure occurs in each canthus. Apical constrictions of amnioserosa cells and contractile actomyosin cables provide forces that favor closure. The two opposing sheets of lateral epidermis that flank the amnioserosa come together in the canthi where they are zipped together. Zipping at the canthi ensures the formation of a continuous epithelium and serves to maintain the curvature of the actomyosin cable necessary to resolve force in a dorsal–ward direction. This dissertation first describes the formation of the canthi, particularly interesting due to the radically different tissue organization for germ band retraction, the preceding stage of development. After canthus ontogeny, I describe dorsal closure stage canthi in three morphologically and mechanically distinct zones. I interrogate each zone by both confocal fluorescent microscopy and laser microsurgery to achieve a thorough visual and mechanical description. Finally, I describe the results of completely removing both canthi — the lateral epidermis leading edges straighten out to become parallel or nearly parallel fronts that move at native or nearly native rates and closure completes at the dorsal midline. Closure, again, proves to be robust and resilient — it can proceed without zipping or much if any leading edge curvature that in control embryos resolves purse string contraction into dorsal–ward forces. In total, the canthus proves to be an excellent source for many avenues of investigation with many more questions left to answer.

Mechanical properties have a decisive role in the fundamental functions of biological systems, including migration of cells, cell apoptosis, and proliferation of cells and bacteria. This is also true for cancer metastasis and morphogenetic processes during embryonic development. It isn’t easy, however, to study biological systems due to their complex behavior, such as their activity and nonlinear material properties. Note that while the individual mechanical properties of specific biological systems, such as biopolymers, have been well established, the collective behavior of these elements has a different response, as the comparative studies of the mechanical properties of single cancer cells and cancerous tissue demonstrate. Thus, numerous experimental instruments have been developed over the years to investigate biological systems’ mechanical properties, individually or collectively. These experimental techniques can evaluate mechanical properties at multiple scales. Theycan target individual biological entities, like single cells, or assess the collective mechanical properties of more complex biological systems, such as tissues or organoids. The resolution of these studies ranges from single-cell analyses to those concerning embryonic morphogenesis. Simulating biological systems’ individual or collective behavior using a discretized approach (i.e., Molecular Dynamics) or a continuum approach (i.e., Finite Element) is an adjunct to experimental studies. This thesis explores the collective behavior observed in individual cells and embryonic tissue. This exploration was carried out through the development of experimental protocols and the application of continuum mechanics models. In the initial two chapters of this thesis, we delve into the fundamental mechanical concepts essential for understanding the mechanical properties of cells and tissues. We also discuss prior studies that employed shell mechanics to model cellular and embryonic deformations. In the third chapter, we detail our collaborative work with Dr. Samaneh Rezvani focuses on the role of the actin cortex in the deformation of individual suspended spherical cells. For this purpose, we utilized double-trap optical tweezers in conjunction with a viscoelastic pressurized-thick-shell model. Using our simulation approach, we determined the mechanical properties of the actin cortex from the experimental results. The elastic shear modulus of the actin cortex ranged between 4.5 kPa and 7.5 kPa. In modeling the steady deformation of single cells with the shell model, we observed that cell volume remains conserved during deformation. Instead of reducing volume, cells extend the actin cortex to accommodate the increased surface area. We also introduced a multilayer viscoelastic shell model to examine the time-dependent mechanical behaviors of cells, focusing on hysteresis due to dissipative processes. Our model incorporated a fluid core within a viscoelastic shell, offering a more thorough understanding of cell mechanics. Our findings indicate that the damping response in cells is predominantly influenced by the viscosity of the cytosol rather than that of the actin cortex. The fourth chapter describes the modeling of experiments conducted by Dr. Renata Garces on gram-negative E. coli bacteria uniaxially compressed between parallel plates. We used Finite Element Modeling (FEM) to examine the collective mechanical behavior of the peptidoglycan layer (PG) in the bacterial cell wall, modeled as a thin, pressurized rod-shaped shell. Finally, in chapter five, we investigated, in collaboration with Dr. Chonglin Guan, the cells’ collective behavior in epithelial tissue during dorsal closure (DC) in developing Drosophila melanogaster embryos (DME). Utilizing glass microprobes, we deformed various tissue types, specifically amnioserosa (AS) and lateral epidermis (LE), and subsequently recorded their responses to assess the impact of tissue mechanical properties on embryonic development. We simulated a viscoelastic flat shell, replicating the geometry of individual embryos, using the Finite Element Method (FEM) to model tissue deformations. Through this methodology, we quantified the mechanical characteristics of amnioserosa and lateral epidermis, encompassing both their viscosity and elasticity. Our analyses determined the elasticity of AS to be approximately (110 to 180 kPa) and its viscosity to be (0.86 to 1.05 Pa.s). Additionally, we executed step-function experiments to ascertain tissue mechanical properties and evaluate tissue relaxation time. Our findings are in line with our previous results obtained from hysteresis studies.

Cell sheet morphogenesis is essential for metazoan development and homeostasis, contributing to key developmental stages such as neural tube closure as well as tissue maintenance through wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. Closure is a remarkably robust process where coordination of conserved gene expression and signaling cascades regulate cellular movements that drive closure. While well-characterized, new ‘dorsal closure genes’ continue to be discovered due to advances in microscopy and genetics. Here, we use live imaging and a set of large deletions, deficiencies (Dfs), that together remove 98.9% of the genes on 2L in order to identify regions of the genome required for normal closure. We successfully screened 96.1% of the genes on 2L and identified diverse dorsal closure defects in embryos homozygous for 47 Dfs, 26 of which have no known dorsal closure gene located within the Df region. We have already identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on the 2L affecting lateral epidermal cell shapes, and anticipate we will continue to identify novel ‘dorsal closure genes’ with further analysis. We also investigate the changes in dorsal closure dynamics and forces in the even-skipped (eve) mutant, which has aberrant cell shapes and behaviors as well as reduced actin and myosin at the purse string, but completes closure. We find that loss of wg/wnt-1 signaling in eve causes the observed defects in closure and that crumbs, a regulator of actin and myosin, is mis-expressed. Additionally, laser microsurgery demonstrates that the eve or wg mutant embryos are under a global tension in the anterior-posterior direction. Lastly, we identify a lesion in echinoid that is responsible for the jagged purse string and ectopic zipping dorsal closure phenotype previously thought to be due to a lesion in Zasp52.

Cell sheet morphogenesis characterizes key developmental transitions throughout phylogeny. As such, it plays a crucial role in developmental milestones in vertebrate morphogenesis including gastrulation, neural tube formation, and palate formation. It also plays important roles in wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis due to the ability to image embryos in-vivo the genetic tractability of Drosophila. While 140 genes are currently published to affect dorsal closure, new genes are identified each year. In addition our understanding of dorsal closure is far from complete with many questions remaining regarding the molecular mechanisms involved in this complex process. To identify a more complete list of genes involved in dorsal closure, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of the 2nd chromosome. Through two crosses, we unambiguously identified homozygous deficiencies and imaged them for the duration of dorsal closure. Images were then analyzed for defects in the cell shapes and morphogenesis. 48 deficiencies were identified to have notable defects on dorsal closure. We anticipate these deficiencies will lead to the identification of at least 31 novel dorsal closure genes. We expect the large number of novel dorsal closure will identify links to pathways already known to coordinate various aspects of closure in addition to new processes and pathways that are currently unidentified as involved in closure.

Cell-cell adhesions and intercellular ion channels, such as gap junctions regulate the synchronization of cells in an epithelium and promote proper embryonic epithelial organization and morphogenesis. Gap junctions regulate the exchange of small molecules and ions, such as calcium (Ca2+) between apposed cells. The strict maintenance of Ca2+ ion concentration across a cell membrane allows Ca2+ to function as an effective intracellular signal in epithelial sheet morphogenesis, including development and wound repair, where it coordinates cell behavior throughout the tissue. Here I used live imaging to observe Drosophila dorsal closure (DC), a model of epithelial sheet morphogenesis. I quantified endogenous calcium flashes in the lateral epidermis, the spread of Ca2+ following single cell wounds, and perturbed extracellular Ca2+ concentrations to demonstrate the necessity of calcium in maintaining the coordinated movements of epithelial sheets in DC. I also genetically and pharmacologically reduced gap junction subunit functionality. I have observed that the knockdown of single ion channel subunits results in the partial penetrance of morphological abnormalities during DC. The intercellular calcium dynamics in the lateral epidermis are modified in gap junction mutants. Lastly, pan-innexin inhibition using gap junction antagonists completely halts DC, resulting in a large coordinated release of intracellular Ca2+ and disruption of cell junction adhesion in the amnioserosa.

Physical forces play a key role in the morphogenesis of embryos. As cells and tissues change shape, grow, and migrate, they exert and respond to forces via mechanosensitive proteins and protein complexes. How the response to force is regulated is not completely understood.

Dorsal closure in Drosophila is a model system for studying cell sheet forces during morphogenesis. We demonstrate a role for mechanically gated ion channels (MGCs) in dorsal closure. Microinjection of GsMTx4 or GdCl₃, inhibitors of MGCs, blocks closure in a dose-dependent manner. UV-mediated uncaging of intracellular Ca2+ causes cell contraction whereas the reduction of extra- and intracellular Ca2+ slows closure. Pharmacologically blocking MGCs leads to defects in force generation via failure of actomyosin structures during closure, and impairs the ability of tissues to regulate forces in response to laser microsurgery.

We identify three genes which encode candidate MGC subunits that play a role in dorsal closure, ripped pocket, dtrpA1, and nompC. We find that knockdown of these channels either singly or in combination leads to defects in force generation and cell shapes during closure.

Our results reveal a key role for MGCs in closure, and suggest a mechanism for the coordination of force producing cell behaviors across the embryo.

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.