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Role of the cytoskeleton in basement membrane-induced mammary epithelial morphogenesis and differentiation

University of British Columbia

Basement membrane-mediated mammary epithelial morphogenesis is characterized by both morphological changes to the cells and the induction of milk protein gene expression. When mammary epithelial cells are placed on a reconstituted basement membrane gel, they aggregate, pull the gel around them and cavitation takes place. During this morphogenic process, the cells undergo changes in shape, polarity and cell-cell junctions. In this thesis I have sought to identify the role of the three major cytoskeletal elements in theses changes and to determine their involvement in the associated induction of milk protein gene induction. When scp2 cells were placed on Matrigel, they formed polarized structures with a central lumen that resemble functional mammary alveolar in vivo. Within these "mammospheres" E-cadherin was localized to adherens junctions, occluding was localized to tight junctions and actin filaments formed an apical junction-associated network. These mammospheres differentiated and expressed two milk proteins: lactoferrin, which is transcriptionally regulated by cell rounding only and β-casein, which is transcriptionally regulated by α6β4 integrin-mediated morphogenic changes initiated by the ligation to laminin within the Matrigel. The cells cultured on Matrigel were treated with increasing concentrations of nocodazole (ND), acrylamide (Ac) and cytochalasin D (CD) for 4 hours and allowed to under go morphogenesis for 72 hours. All three disrupting agents prevented the cells from undergoing morphogenesis and inhibited differentiation. ND treatment inhibited the synthesis of both lactoferrin and fi-casein while Ac and CD treatments inhibited the β casein induction only. These observations suggested that ND was not specifically inhibiting differentiation by preventing morphogenesis. In contrast, Ac and CD appeared to inhibit differentiation in a morphogenesis-dependent manner. The α6β4 integrin becomes physically linked to keratin intermediate filaments after its ligation to laminin. Because α6β4 ligation also initiates apical/basal polarity, cells on Matrigel were treated with a short term high-dose of CD to completely disrupt factin and were then observed for a six day recovery period. The apical actin network and tight junctions were re-established first followed by the induction of β-casein. Therefore, keratin and actin appear to influence functional mammosphere formation by helping to initiate and maintain cell polarity.

Thesis/Dissertation

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2009-06-29

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http://hdl.handle.net/2429/9780

Anatomy, Cell Biology and Physiology

University of British Columbia

UBCV

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