The Shroom family of proteins is currently comprised of four members, Shroom1, 2, 3 and 4. Since Shroom3 was shown to be a critical protein for neural tube closure, the other three proteins are also expected to play an important role for proper development. However, their functions during development were not clear. To address this, my study started with Shroom3 function in the neural plate. Shroom3 had been previously known to induce apical constriction by controlling actin filaments in neuroepithelial cells. My studies show that Shroom3 induces apico-basal cell heightening by controlling parallel microtubule assembly. Shroom3 is able to change the distribution of γ-tubulin, suggesting that Shroom3 controls apical constriction and apico-basal cell elongation via both actin filaments and microtubules. The ability to control γ-tubulin distribution is possessed not only by Shroom3, but also by all other Shroom proteins, although they can not induce apical constriction. In addition, they are expressed in tissues which contain apico-basally elongated cells. Data from functional assays with Shroom2 show that it induces cell elongation and is required for proper cell shape in deep layer neuroepithelial cells in Xenopus. These data suggest that Shroom family proteins control cell architecture during morphogenetic development. I have discovered another role for Shroom2. By comparative analysis with Xenopus and Physalaemus, which have different pigment patterns in eggs, I show that a high level of maternal Shroom2 mRNA is important for pigment polarity in Xenopus. Furthermore, Shroom2 controls the distribution of spectrin which plays a role in pigment granule movement. Thus, Shroom2 is suggested to be a key molecule to control the pigment polarity in amphibian eggs. Together all these data suggest that Shroom family proteins play a role in cell morphogenesis and polarization via controlling the cytoskeleton during Xenopus development. / text

The bacterial cell wall, composed of peptidoglycan (PG), is an essential component of the cell envelope. This macromolecular structure fortifies the cell membrane, determines cell shape, and helps prevent osmotic lysis. The synthesis and remodeling/recycling of this polymer is mediated by PG synthases and hydrolases, respectively. Proper control of the PG hydrolases is particularly important since misregulation of these enzymes can lead to lethal breaches in the cell wall. Surprisingly, however, the precise molecular mechanisms governing the activities of these enzymes remain poorly understood. To help understand how PG hydrolases are regulated, I examined how their activity is controlled during cytokinesis in Escherichia coli. One important class of PG hydrolases necessary for cell division is the LytC-type amidases (AmiA, AmiB and AmiC). These enzymes require activation by the LytM factors EnvC and NlpD. My work focused on elucidating the mechanism by which the LytM factors activate the amidases. Using a genetic enrichment strategy, I isolated amiB misregulation mutants. Interestingly, the mutations mapped to a region of AmiB found only in cell separation amidases. Structural analysis of an AmiB ortholog indicates that this region corresponds to an alpha-helical domain that appears to occlude the active site. Thus, activation of the amidases by the LytM factors likely occurs via a conformational change that displaces the regulatory helix from the active site. In addition to amidase regulation, I also investigated how the LytM activators are recruited to, and regulated at the site of division. Using genetic and biochemical approaches, I showed that EnvC is directly recruited to the division site by FtsEX, an ATP-binding transporter- like complex. Interestingly, ATPase-defective FtsEX derivatives can still recruit EnvC to the divisome, but fail to promote cytokinesis. These results support a model where conformational changes induced by the ATPase activity of FtsE are directly and specifically transmitted to the amidases via FtsX and EnvC. This model is attractive because it provides a mechanism for converting the potentially dangerous activity of septal PG splitting into a discrete process which can be cycled on and off in coordination with the division process.

Patterning, morphogenesis, and cell divisions are distinct processes during development yet are concurrent and likely highly integrated. However, it has been challenging to investigate them as a whole. Recent advances in imaging and labeling tools make it possible to observe live tissues with high coverage and resolution. In this dissertation work, we developed a novel imaging platform that allowed us to fully capture the early neural tube formation process in live zebrafish embryos at cellular resolution. Importantly, these datasets allow us to reliably track single neural progenitors. These tracks carry information on the history of cell movement, shape change, division, and gene expression all together. By comparing tracks of different progenitor fates, we found they show a spatially noisy response to Sonic hedgehog (Shh) and become specified in a positionally mixed manner, in surprising contrast to the "French Flag" morphogen patterning model. Both cell movement and division contribute to cell mixing. In addition, we decoupled the temporal and genetic regulatory network (GRN) noises in Shh interpretation using tracks that carry both Shh signaling and cell fate reporters. Our tracks suggest that, after specification, progenitors undergo sorting to self-assemble a sharp pattern. Consistent with this hypothesis, we found ectopically induced progenitors move to correct locations. Furthermore, we show that proper adhesion is required for cell sorting to happen (Chapters 2 and 3). In the cleavage stage embryos, the cells on the surface undergo shape changes followed by lineage separation and differentiation. We quantitatively measured this morphogenesis process and tracked cell divisions. By applying a mathematical model we uncover a predictive, and perhaps general link between cell division orientation, mechanical interaction, and the morphogenetic behavior of the whole surface layer (Chapter 4). Finally, we discuss the concepts and tools of cell tracking including a multi-color cell labeling method we developed by modifying the "Brainbow" system (Chapter 5). Together this dissertation showcases the importance and promise of live observation based, quantitative and integrated analysis in our understanding of complex multi-cellular developmental processes.

The fascinating capability of cellular self-organization during tissue development and repair is a central question in developmental biology and regenerative medicine. Understanding the dynamic morphogenic and regenerative processes of biological tissues will have important implications in biology and medicine. Nevertheless, the elucidation of the cellular self-organization processes is hindered by a lack of effective tools for monitoring the spatiotemporal gene expression distribution and a lack of ability to perturb the self-organization processes in living cells and tissues. Multimodal modularities that allow both single cell perturbation and gene detection are required to enable a new paradigm in the investigation of complex tissue morphogenic processes. To address this critical challenge in the field of developmental and regenerative medicine, we are developing a multimodal gold nanorod-locked nucleic acid (GNR-LNA) composite for single cell gene expression analysis in living cells and tissues at the transcriptional level. Using antisense RNA sequences, we design LNA probes for detecting specific molecular targets in living cells. The LNA probes bind to the GNR spontaneously due to the intrinsic affinity between the GNR and LNA. In close proximity, the fluorescent probes are effectively quenched by the GNR. Therefore, a fluorescent signal is only observed when the specific target thermodynamically displaces the LNA probe from the GNR. Furthermore, the GNR also serves as a transducer for photothermal ablation. Thus, we established a novel modularity for imaging the spatiotemporal gene expression distribution in living cells and tissues. The single cell analysis capability of our techniques enables us to adopt a unique approach to study the tissue regenerative processes during normal development and diseases, and this will have a profound impact on regenerative medicine and disease treatment in future. Moreover, we applied this GNR-LNA probe to explore the endothelial cell mRNA dynamics during capillary morphogenesis. Three different types of cells were identified due to their different roles during endothelial cell capillary-like formation process. Our findings indicated that the endothelial cell behavior is directly related to the Dll4 mRNA expression, and Dll4 expression in ECs determine the cell fate. Our GNR-LNA probe enable us to investigate the correlations between Dll4 mRNA expression and cell behavior during capillary morphogenesis. Experimental results indicated that: (1) When the endothelial cells aggregate, the cells migrate with certain displacement, the Dll4 mRNA expression decreases. (2) When the endothelial cells sprout, the cells migrate with small displacement but the cell shape changes to an ellipse shape, the Dll4 mRNA expression begin to increase. (3) When the endothelial cells elongate and form cell-cell contract with adjacent cells, the Dll4 expression decreased to a certain level and keep stable until the cell activity change to another stage. Furthermore, it has been demonstrated endothelial cells compete for the leader cell position during wound healing, collective cell migration, and tip cell formation during angiogenic process. It has been demonstrated that endothelial cells compete for the tip cell formation through Notch signaling pathway. However, how the mechanical force regulates tip cell formation is still unclear, and if mechanoregulation of tip cell formation through Notch pathway still unknown. Mechanical and chemical regulations of tissue morphogenesis and angiogenesis are being investigated in both in vitro capillary-like network formation assay and in vivo mice retina angiogenesis assay. Here, we investigated the mechanoregulation of mechanotransduction of tissue morphogenesis and angiogenesis using both in vitro endothelial cell tube formation model and in vivo mice retina blood vessel development model. Our results demonstrated that (1) Notch pathway negatively regulates tip cell formation: inhibition of Notch pathway (DAPT) enhances tip cell formation, induces Dll4 and Notch1 activity, activation of Notch pathway (Jag1 peptide) inhibits tip cell formation, suppresses Dll4 and Notch1 activity. (2) Mechanical force negatively regulate tip cell formation: (a) Decrease mechanical force via Rho kinase inhibitor Y-27632, myosin II inhibitor Blebbistatin, or laser ablation, enhances tip cell formation and induces Dll4 activity through mediation of Dll4-Notch1 lateral inhibition, (b) increase mechanical force via traction force inducer Nocodazole and Calyculin A, suppresses tip cell formation and inhibits Dll4 activity through activation of Notch pathway. (3) Mechanical force negatively regulates tip cell formation partially via mediation of Notch pathway. Mechanical force is necessary for tip cell formation and negatively regulate tip/stalk selection via Dll4-Notch1 lateral inhibition. Interruption of mechanical force enhance tip cell formation via suppression of Dll4-Notch1 lateral inhibition, thus resulting the increase of Dll4 expression. Enhance of mechanical force inhibits tip cell formation via activation of Dll4-Notch1 lateral inhibition, thus resulting the decreases of Dll4 expression. All these finding wills have great significance for various biomedical applications, such as tissue engineering, cancer, and drug screening.

Magistrantūros studijų baigiamajame darbe pateikiama 2011–2012 metais Aleksandro Stulginskio universiteto Agronomijos fakulteto Agrobiotechnologijos laboratorijoje tirtų veiksnių, lemiančių sėjamosios kanapės dediferenciacijos indukciją hipokotilių ir skilčialapių kultūroje, tyrimų rezultatai. Darbo objektas – 10 dienų eksplantai auginti MS terpėje su skirtingais augimo reguliatorių kiekiais. Darbo metodai: Sėjamosios kanapės sėklos plautos po tekančiu vandeniu 1 val., 15 min. sterilintos 0,2 % kalio permanganato tirpale, 2 min. laikytos 70 % etanolio vandeniniame tirpale, 9 min. 10 % natrio hipochlorite ir 3 kartus po 5 min. mirkytos distiliuotame vandenyje. Sterilios sėklos daigintos ant MS maitinamosios terpės be augimo reguliatorių, papildytoje 10 g l-1 sacharozės ir 8 g l-1 Difco-Bacto agaru. MS terpėje su skirtingais augimo reguliatorių kiekiais auginta po 60 eksplantų. Kas keturias savaites eksplantai perkelti į šviežią tos pačios sudėties maitinamąją terpę. Vertintas kaliaus susidarymo dažnis (%), šaknų formavimosi dažnis (%), šaknų kiekis iš eksplanto (vnt.) bei ūglių formavimosi dažnis (%). Darbo rezultatai. Įvertinus sėjamosios kanapės kaliaus indukcijos tyrimų rezultatus nustatyta, kad sėjamosios kanapės izoliuoti hipokotilio audiniai nedideliu dažniu geba formuoti kalių ir terpėje be augimo reguliatorių. Naudojant vienos grupės (auksiną arba citokininą) augimo reguliatorių didžiausias kaliaus formavimosi dažnis gautas terpėje, papildytoje 0,5 mg l-¹ α... [toliau žr. visą tekstą] / The master work presents the results of factors affecting Cannabis sativa L. dedifferentiation process in vitro. Research was investigated at the Laboratory of Agrobiotechnology, Faculty of Agronomy of Aleksandras Stulginskis University in 2011 - 2012. Investigation in vitro was carried out with Cannabis sativa L. hypocotyls and cotyledons explants. Object of the work – 10 days explants grown in MS medium with different growth regulators concentrations. Method of the work – Cannabis sativa L. seeds were washed under running water for 1 h and surface sterilized with 0.2 % potassium permanganate for 15 min. sterilized and for 2 min. in 70 % ethanol, then sterilized in 10 % sodium hypochlorite for 9 min. and rinsed 3 times for 5 min. with sterile distilled water. Sterile seeds sprouted on MS nutrient medium without growth regulators, supplemented with 10 g l-1 sucrose and 8 g l-1 Difco Bacto-agar. MS medium with different growth regulators concentrations were grown 60 explants. Every four weeks, explants were transferred to the same composition fresh medium. The callus formation frequency (%), root formation frequency (%), root number of the explant (units) and the frequency of shoot formation (%) were evaluated. The results of the work – Cannabis sativa L. hypocotyls tissues with low frequency can form callus and without growth regulators in the medium. Using a single group of growth regulators (auxin or cytokinin) highest callus formation frequency obtained in the medium... [to full text]

Magistrantūros studijų baigiamajame darbe pateikiami veiksnių, lemiančių pluoštinių linų morfogenezę in vitro, tyrimų duomenys. Darbo objektas – keturios pluoštinių linų veislės 'Dangiai', 'Sartai', 'Snaigiai' ir 'Vaižgantas'. Darbo metodai. Pluoštinių linų morfogenezei somatinių audinių kultūroje tirti naudoti hipokotilio eksplantai. Eksplantai auginti MS terpėje su skirtingais augimo reguliatorių kiekiais auginimo kambaryje. Vertintas pridėtinių pumpurų formavimosi dažnis (%) ir pridėtinių ūglių kiekis iš eksplanto (vnt.). Darbo rezultatai. Egzogeninių augimo reguliatorių priedas indukcijos terpėje daugeliu atvejų skatino tirtų pluoštinių linų veislių ūglių regeneraciją, tačiau optimali šio junginio koncentracija priklausė nuo genotipo. Iš tirtų pluoštinių linų veislių didžiausia morfogenine galia pasižymėjo veislės 'Vaižgantas' izoliuoti hipokotiliai. Priklausomai nuo genotipo ir maitinamosios terpės sudėties pluoštinių linų pridėtinių pumpurų formavimosi procesas hipokotilių kultūroje vyko 2,87-94,50 % dažniu regeneruojant 1,67-6,00 ūglius iš eksplanto. Efektyviam veislės 'Dangiai' ūglių regeneravimui maitinamąją terpę tikslinga papildyti 2,0 mg l-1 BAP + 0,1 mg l-1 NAR bei eksplantus paveikti 4 °C temperatūra 24 valandas. Veislės 'Sartai' izoliuotų hipokotilių audinių antrinę diferenciaciją skatino 72 valandų 7 °C temperatūros poveikis bei citokinino 1,0 mg l-1 2iP priedas maitinamojoje terpėje. Intensyviausiai veislės 'Snaigiai' eksplantai pumpurus formavo maitinamojoje... [toliau žr. visą tekstą] / The master work presents the results of fibre flax morphogenesis in vitro affecting factors. Object of the research − four fibre flax cultivars 'Dangiai', 'Sartai', 'Snaigiai' and 'Vaižgantas'. Research methods. To study fiber flax morphogenesis of somatic tissues hypocotyls were used as explant. Explants were placed on MS medium supplemented with different combinations of growth regulators and cultivated in growth chamber. The percentage of bud regeneration and the number of shoots per explant were evaluated. Research results. Exogenous growth regulators concentration in induction media increased fiber flax shoots regeneration, and therefore specific combination of growth regulators must be designed for each genotype. Among investigated genotypes, the hypocotyls of cultivars 'Vaižgantas' manifested the best morphogenic capability. Depending on genotype and medium composition fiber flax adventitious buds regeneration frequency varied from 2.87-94.50% and the number of shoots per explant reached 1.67-6.0, respectively. Hypocotyls of cultivar 'Dangiai' showed the best response on medium supplemented by 2.0 mg l-1 BAP with 0.1 mg l-1 NAA and pretreated by 4 °C temperature 24 hours. In the presence of only cytokinins, the highest mean value of regenerated shoots was observed on medium supplemented with 1.0 mg l-1 2iP and 72 hours 7 °C temperature pretreatment for hypocotyls of 'Sartai'. Growth regulators combination 2.0 mg l-1 2iP with 0.1 mg l-1 NAA and treatment by 4 °C... [to full text]

Magistrantūros studijų baigiamajame darbe pateikiami sėjamosios kanapės eksplanto tipo ir maitinamosios terpės sudėties poveikio mofogenezei in vitro tyrimų rezultatai. Darbo objektas – sėjamosios kanapės stiebo ir lapkočio segmentai. Darbo metodai: Sėjamosios kanapės eksplantai auginti MS, B5 ir White maitinamosiose terpėse, papildytose įvairiais augimo reguliatorių deriniais. Vertintas kaliaus susiformavimo dažnis (%), ūglių susiformavimo dažnis (%) ir šaknų susiformavimo dažnis (%). Darbo rezultatai. Sėjamosios kanapės morfogenezės procesas didžiąja dalimi priklauso nuo bazinės maitinamosios terpės sudėties. Izoliuoti stiebo segmentai intensyviausiai kalių indukavo bazinėje B5 maitinamojoje terpėje, o lapkočio segmentai − B5 maitinamojoje terpėje, papildytoje 1,0 mg l-1 TDZ + 0,5 mg l-1 NAR arba 1,0 mg l-1 KIN + 0,5 mg l-1 NAR. Pridėtinių ūglių formavimasis intensyviausiai vyko bazinėje B5 maitinamojoje terpėje, papildytoje 1,0 mg l-1 TDZ + 0,5 mg l-1 NAR (iš stiebo segmento audinių) ir 1,0 mg l-1 KIN + 0,5 mg l-1 NAR (iš lapkočio audinių). Izoliuoti stiebo segmentai didžiausiu dažniu šaknis formavo B5 maitinamojoje terpėje, papildytoje 1,0 mg l-1 KIN + 0,5 mg l-1 NAR, o lapkočio segmentai − 1,0 mg l-1 TDZ + 0,5 mg l-1 NAR. Tirti stiebo segmentai pasižymėjo didesne geba formuoti kalių, pridėtinius ūglius ir šaknis nei izoliuoti lapkočio segmentai. / The master work presents the effect of explants type and medium composition on morphogenesis in vitro of hemp. Object of the research – stem segments and leafstalk of hemp. Research methods. To study hemp morphogenesis in vitro hypocotyls and leafstalk were used as explants. Explants were placed on MS, B5 and White media supplemented with different combinations of growth regulators. The percentage of callus formation, shoot induction and root formation were evaluated. Research results. The current study indicates that there is strong medium composition effect on morphogenesis in vitro of hemp. From tested media most intensive callus genesis was observed on B5 medium, but from leafstalk – on B5 medium with 1.0 mg l-1 TDZ + 0.5 mg l-1 NAA or 1.0 mg l-1 KIN + 0.5 mg l-1 NAA. The growth regulators combination 1.0 mg l-1 TDZ + 0.5 mg l-1 NAA on B5 media was the most suitable for stem segments shoots induction while growth regulators combination 1.0 mg l-1 KIN + 0.5 mg l-1 NAA promoted the highest frequency of shoots induction from leafstalk segments. The highest root formation frequency was observed on the same basal medium supplemented with 1.0 mg l-1 KIN + 0.5 mg l-1 NAA from stem segments, but from leafstalk – on B5 medium with 1.0 mg l-1 TDZ + 0.5 mg l-1 NAA. Generally, the stem segments exhibited a higher morphogenic response than leafstalk of hemp.

Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

<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 <italic>retinitis pigmentosa</italic>. 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. </p><p>In <italic>Drosophila melanogaster</italic> mutations in MyoVIIa encoded by <italic>crinkled (ck)</italic> cause defects in hair and bristle morphogenesis and deafness. Here we study the formation of bristles and hairs in <italic>Drosophila melanogaster</italic> 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.</p><p>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.</p><p>We further demonstrate that over-expression of <italic>Quail</italic> (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 <italic>Alpha-actinin-GFP</italic>, another actin bundling protein, phenocopies the multiple hair phenotype of ck/MyoVIIa over-expression. Over-expression of <italic>Alpha-actinin-GFP</italic> in a ck/MyoVIIa null or near null background shows that <italic>Alpha-actinin-GFP</italic> cannot rescue the split or short hair phenotype of ck/MyoVIIa loss-of-function. However, cells over-expressing <italic>Alpha-actinin-GFP</italic> in a ck/MyoVIIa null or near null background have more than the normal 8-10 split hairs, suggesting that <italic>Alpha-actinin-GFP</italic> 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 <italic>Twinfilin</italic>, 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. </p><p>In addition, we use yeast two-hybrid strategies to identify <italic>Slam</italic> as a protein that directly binds to ck/MyoVIIa. We show that <italic>Slam</italic>, a novel membrane-associated protein, likely functions to regulate ck/MyoVIIa function during hair and bristle morphogenesis. We show that over-expression of <italic>Slam</italic> and loss-of-function mutations in <italic>Slam</italic> phenocopy ck/MyoVIIa loss-of-function split and short hair phenotype. We also show that disruption of <italic>Slam</italic> and <italic>RhoGEF2</italic> association causes split hair defects similar to ck/MyoVIIa loss-of-function phenotype suggesting that Slam probably regulates ck/MyoVIIa function via <italic>RhoGEF2</italic>.</p><p>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.</p> / Dissertation