Terry Lechler - US grants

DESCRIPTION (provided by applicant): Desmosomes are essential cell-cell adhesion structures that provide mechanical integrity to tissues. Disruption of desmosomes leads to a range of diseases in man including skin fragility and blistering disorders as well as cardiomyopathies. The textbook view of desmosomes is that they are rather passive structures that simply bind to the intermediate filament cytoskeleton. We are now beginning to appreciate that desmosomes are both more dynamic and have more functions than previously thought. For example, we have shown that desmosomes control microtubule organization in the epidermis and cultured keratinocytes. In this application, we focus on how desmosomes control the organization of cytoskeletal networks. Our central hypothesis is that the desmosome actively organizes both intermediate filaments and microtubules through recruitment of a protein complex that is usually found at the centrosome. Some of the novel desmosomal proteins in this complex, including Lis1 and NDEL1, are known to control microtubule and intermediate filament organization in other tissues. By understanding both how these proteins are recruited to desmosomes and their functional role there, we will greatly expand our knowledge of how desmosomes control the cytoskeleton. This will allow us to specifically disrupt microtubule organization in differentiated epidermis, and to determine the physiological function of cortical microtubules in these cells. To accomplish this, we will generate and characterize mouse models in which centrosomal proteins are not recruited to desmosomes. This will directly test the physiological role of this novel desmosomal function. Second, we will determine how loss of Lis1 in the epidermis leads to defects in epidermal integrity, desmosome architecture, and microtubule organization. Third, we will determine the role of the novel keratin-binding protein, NDEL1, in controlling keratin filament organization downstream of the desmosome. This work will lead to a mechanistic understanding of how desmosomes reorganize the underlying cytoskeleton and to a greater understanding of why desmosome disruption results in such a diversity of pathological phenotypes. PUBLIC HEALTH RELEVANCE: Desmosomes are cell-cell adhesion structures that provide mechanical integrity to cells by linking to the underlying cytoskeleton network. Disruption of desmosomes causes epidermal fragility, skin blistering, and cardiomyopathies. We will characterize novel mechanisms that desmosomes use to reorganize the cytoskeleton. This will allow an understanding of how desmosome disruption results in such a diversity of pathological phenotypes.

DESCRIPTION (provided by applicant): During embryonic development, a single-layered ectodermal cell layer transforms into a multi- layered epidermis. The multi-layered or stratified architecture of the epidermis is essential for its barrier function - allowing it to prevent both dehydration and infection. Stratification of the epidermis is driven by asymmetric cell divisions that generate one proliferative basal cell and one suprabasal cell that expresses differentiation markers. We want to understand how epidermal cells reorient their mitotic spindle in order to divide asymmetrically. A necessary first step toward this goal is to define the dynamics of both spindle poles and microtubules during the process of spindle reorientation. We have developed the tools and methodologies to complete this analysis. On the molecular level, NuMA is a core component of the asymmetric cell division machinery. In invertebrates, it is required for asymmetric cell divisions, though how it is localized and what its function is remains undetermined. We will test the functional relevance of NuMA's protein-protein interactions in both its localization to the cell cortex and in spindle reorientation. The work proposed will have broad significance as asymmetric divisions play fundamental roles in cell-type diversification, stem cell homeostasis, morphogenesis and the immune response. Additionally, disruption of asymmetric divisions has been proposed to promote tumorigenesis. Therefore, understanding the mechanisms driving asymmetric cell division will provide insights not only into epidermal stratification but also additional physiological and pathological processes.

DESCRIPTION (provided by applicant): Differentiation induces a change in microtubule organization in cell types as diverse as epithelia, muscles and neurons. This requires loss of the centrosome's microtubule organizing activity and formation of non- centrosomal microtubule arrays. We have a very limited understanding of how this reorganization occurs and the functions of the resulting microtubules. In part, this is due to lack of tools to observe and specifically perturb microtubule organization in tissues. The long-term goal of our work is to understand how diverse microtubule organizations are generated in differentiated cells and to expose their functions. The objectives of this proposal are to determine how loss of centrosomal organizing activity and microtubule minus end anchoring proteins collaborate to induce specific microtubule arrays. Based on our preliminary data, we hypothesize that differentiation causes changes in both nucleation and anchoring of microtubules at centrosomes. In combination with cytoplasmic minus end anchoring proteins, this allows for specific cytoskeletal structures to form in differentiated cells. We have developed tools/technologies that will allow us to fully characterize the changes in centrosome nucleation activity, ultrastructure and protein composition that occur upon differentiation in the epidermis. In addition, we will elucidate the underlying molecular mechanisms that lead to changes in centrosome composition and activity. Finally, we have developed novel mouse lines that will be used to examine the roles of noncentrosomal microtubule arrays in both the skin and the intestine through specific disruption of microtubule minus end anchoring proteins. These studies will elucidate the mechanisms regulating centrosome activity and microtubule reorganization in differentiated cells and determine the physiologic functions of these arrays in two different tissues.

? DESCRIPTION (provided by applicant): Desmosomes are cell-cell adhesion structures that are required for the mechanical strength of the epidermis. Disruption of desmosomes results in devastating effects on skin integrity in diseases including pemphigus vulgaris/foliaceus and some types of epidermolysis bullosa. Mutations in desmosomal proteins also lead to cardiomyopathies/dysplasias. Our long-term goal is to understand the assembly and functions of desmosomes and the cellular responses to their disruption. The canonical function of desmosomes is to physically link keratin networks between cells. While we have a good understanding of the direct physical interactions that mediate keratin attachment, it is not known whether desmosomes affect keratin assembly. We have identified a novel desmosomal protein with the unique ability to promote the local assembly of keratin filaments. This changes our view of desmosomes from relatively passive structures that simply bind cytoskeletal networks to active modulators of their assembly. We will study the mechanism and regulation of this remarkable activity, which is expected to identify a novel pathway for desmosome stability and epidermal integrity. Our second aim will use a targeted proteomics approach to identify novel and transiently associated desmosomal proteins. This will identify additional putative disease genes and allow future quantitative determination of changes in desmosome composition in various disease states. Finally, our third aim addresses cellular responses to loss of desmosomes. We have found that desmosome disruption leads to dramatic changes in adherens and tight junction activity through effects on protein expression and localization. We will determine how cells sense desmosome disruption and determine the signaling pathways that control alterations of other cell adhesion structures. In total, these studies will increase or understanding of normal desmosome function and cellular responses to their perturbation which we expect to yield diagnostic and therapeutic tools for desmosome-related diseases.

? DESCRIPTION (provided by applicant): Cell division orientation must be tightly controlled for the generation of normal tissue architecture. In the skin, asymmetric cell divisions that are driven by oriented mitotic spindles promote both the stratification and differentiation of the embryonic epidermis. While some of the proteins required for spindle orientation have been identified, we do not fully understand how this molecular machinery generates forces on astral microtubules to allow precise control of cell division orientation. In addition, the essential requirement of spindle orientation in embryonic epidermal development has prevented analysis of the function of these divisions in adult skin. Roles for spindle orientation in the morphogenesis of hair follicles, which are highly organized epidermal appendages, have not been addressed. Similarly, whether spindle orientation is required for homeostasis of the adult interfollicular epidermis has not been tested. This is despite long- standing hypotheses that loss of spindle orientation could promote tumorigenesis. Our preliminary studies have identified an unexpected mechanism required for spindle orientation in keratinocytes which we will elucidate in detail in Aim 1. These mechanistic studies allowed us to generate a mutant mouse model in which spindle orientation was randomized in the epidermis, resulting in neonatal lethality. Importantly, disruption of spindle orientation in adult animals caused both severe hair follicle defects and interfollicular hyperproliferation and local invasion. We will use this new genetic model to determine the specific roles for spindle orientation in hair follicle morphogenesis (Aim 2) and adult interfollicular epidermal homeostasis and tumorigenesis (Aim 3). Together, these data will provide a deeper mechanistic understanding of spindle orientation which has essential functions in epidermal, cardiac and neural development as well as the adaptive immune response. In addition this work has direct relevance for both understanding and eventually treating alopecias/hair follicle disorders and skin cancer.

Abstract During embryonic development, the epidermis transforms from a single cell layer to a stratified, fully differentiated tissue that forms a barrier to the external environment. Stratification is driven by proliferation in both the basal cell layer and in a transient transit-amplifying cell population called intermediate cells. These cells express differentiation markers yet remain mitotic, and are hypothesized to promote tissue expansion. However, the roles for intermediate cells have never been directly addressed. Neither do we understand how these cells remain proliferative in an environment that normally promotes differentiation. Here we take advantage of novel mouse lines that we have developed to address roles of intermediate and spinous cells in the expansion and morphogenesis of the epidermis, and to determine how these cells remain mitotically active when not attached to the underlying basement membrane. These studies are expected to identify pathways to promote tissue expansion/maturation for pre-term infants and for regenerative medicine, as well as provide novel mechanistic basis for signaling between epidermal cell layers.

Abstract The intestine has a simple repeating architecture that is essential for its function. Absorptive villi are compartmentalized from the intestinal crypts, which house the progenitor cells and function as the stem cell niche. Recent studies have made inroads into understanding how villi form embryonically, but the cell biological mechanisms that drive the postnatal morphogenesis of crypts are not known. We have performed morphometric and dynamic transcriptomic analysis of crypt morphogenesis that has led to a framework for understanding this process. In this proposal we focus on three key stages of crypt formation and function. First, we address how the crypts become morphologically compartmentalized from the villi. Our preliminary data demonstrate that this is essential for villar patterning and maximizes surface area for absorption. Second, we analyze how crypts expand uniaxially along the crypt/villus axis in order to package progenitor cells required for the rapid turnover of the gut. Finally, as cells exit the crypt, most must become absorptive enterocytes. We will analyze functions of transcriptional regulators of enterocytes that play important roles in cell fate and tissue architecture. Together, these studies will establish how the intestinal stem cell niche forms and define roles for the stereotypical architecture in stem cell function, cell fate decisions, and tissue function.