Molecular Mechanisms Regulating Cell Polarization and Protein Targeting

My general research interest is to understand the mechanisms of cell polarization and the functional consequences of this polarization during development and cancerogenesis. In the last several years my laboratory has focused on several distinct but related projects: regulation and function of asymmetric cell division, mechanisms of cancer metastasis and regulation of epithelia polarization and cell migration in vitro and in vivo. The latest findings and future directions for these projects are described below. 

1. Molecular mechanisms of epithelial cells polarizatio and apical cilia formation.

Epithelial cells are structurally and functionally polarized to transport specific molecules while maintaining a trans-epithelial barrier. This cellular asymmetry is essential for the proper functioning of epithelial tissues and depends on polarized endocytic transport routes. Additionally, epithelial cells coordinate their polarization with neighboring cells to form an apical lumen, a key step in the establishment of renal and gut architecture, and thereby function. Indeed, malfunctions in epithelial cell polarization and apical lumen formation are responsible for a variety of renal and intestinal disorders, such as polycystic kidney disease, renal tubular acidosis, microvilli inclusion disease and diabetes insipidus. This project aims to fill critical gaps in our understanding of epithelial cell biology, including what are the molecular mechanisms mediating coordinated formation of apical lumen during epithelia morphogenesis and function.

            Despite recent advances in our understanding of the mechanisms mediating lumen formation, many questions remains unanswered. How are endosomes targeted during apical lumen formation? How do cells establish the site of single apical lumen? Do the mechanisms of lumen formation established in vitro also apply to epithelial morphogenesis in vivo? Addressing these pivotal and unanswered questions forms the backbone of this project. This study is designed to analyze the machinery of apical lumen formation using in vitro (MDCK and Caco-2 cell three dimensional cultures) and in vivo (zebrafish; collaboration with Bruce Appel's lab) models.

2. Regulation breast cancer metastasis.

Remodelling of the extracellular matrix (ECM) is a key process during tumor growth and metastasis and is mediated via formation of structures, known as invadopodia, and targeted secretion of enzymes, known as matrix metalloproteinases (MMPs). Invadopodia are actin-rich structures that degrade the ECM and have been shown to be important for tumor progression. MMP2/9 also have been shown to be enriched at the invadopodia and are known for their roles in breast tumor growth/metastasis. As a result, MMP2/9 have emerged as possible therapeutic targets. Unfortunately, clinical trials directly inhibiting MMP2/9 have proven ineffective, mostly due to adverse side effects and pre-existing high levels of secreted MMPs. It has been proposed that targeting the machinery specifically mediating MMP secretion at cancer cell invadopodia is more effective approach. However, little is known about targeted MMP2/9 secretion since most of the studies have focused on the mechanisms mediating ECM and cell adhesion and actin dynamics during metastasis. In contrast, very few studies have analysed the machinery mediating targeted MMP secretion in cancer cells in vitro and tested these mechanisms in vivo. The main goal of this project is to combine studies using basic cell and molecular biology techniques with more translational in vivo approaches to define and systematically analyse novel pathways that mediate targeted MMP transport and secretion during breast cancer metastasis. The strength of this project is combining of the unique expertise from my lab (cell biology and Rab GTPases) and Tracy Lyon's (breast cancer and xenograft assays) laboratories. This combination will allow us to comprehensively test these hypotheses in vitro and in vivo.

3. The role of Rab GTPases in regulating cell migration during development.

Rab-dependent membrane and protein transport plays a key role in many cellular processes, including cell migration. Recently we identified Rab40b as a majopr regulator of cell migration in vitro. Rab40b appears to work by mediating the targeting and activation of MMPs as well as RhoG-Rac1 pathways, thus directly affecting the formation of actin-driven membrane extensions required for cell migration through extracellular matrix. The main goal of this project is to dissect the machinery regulating localized actin dynamics invasive pseudopod extension during cell migration. To that end, we will use various in vitro migration and invasion assays as well as zebrafish neural crest migration model (collaboration with Bruce Appel and Kristin Artinger).

4. The role of midbodies in regulating cancer and stem cell proliferation.

Cytokinesis is the final stage of the cell cycle resulting in physical separation of daughter cells. At late mitosis, the mother cell divides by the formation of a cleavage furrow, leaving two daughter cells connected by a thin intercellular bridge (ICB). The resolution of this bridge, abscission, is mediated by coordinated action between specialized recycling endosomes and cytoskeleton. During ingression of the cleavage furrow, the central spindle microtubules are compacted to form the structure known as the midbody (MB). The MB is situated within the ICB, with the abscission usually occurring at one side of the MB. As a result of this one-sided (asymmetric) abscission, only one daughter cell inherits the post-mitotic MB. These post-mitotic MBs can then either accumulate in the cytoplasm or be degraded. Interestingly, recent studies have identified post-mitotic MBs as novel signaling platforms regulating stem cell fate and proliferation. Indeed, stem cells were shown to accumulate post-mitotic MBs and the induction of MB accumulation leads to reprogramming of cell fate and conversion to highly-proliferative stem cell-like phenotypes. Despite the importance of MBs in determining cell fate and proliferative capacity, the mechanisms that regulate asymmetric MB inheritance and post-mitotic degradation remains completely unknown. As the result, the main goal of this project is to identify the factors mediating MB inheritance and accumulation, and to test the role of these factors in regulating stem cell fate and differentiation.

University of Colorado Anschutz Medical Campus

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