Role of calmodulin-like protein and myosin-10 in epithelial differentiation

Micrograph of a HeLa cell expressing green fluorescent protein (GFP) tagged myosin-10 together with calmodulin-like protein. Note the abundant fluorescent myosin-10 puncta in filopodial extensions. Image taken by Richard D. Bennett (Mayo Graduate School PhD student, Molecular Biology track)

Ca2+ signaling is essential for cell function. Transmission of the Ca2+ signal occurs via Ca2+-sensor proteins such as calmodulin and related members of the Ca2+ binding protein family. Loss or abnormal expression of these proteins correlates with cellular and growth abnormalities observed in cancer cells. However, the mechanism of action of most of these Ca2+ binding proteins remains poorly understood. The objective of this project is to investigate the role in cell migration and differentiation of the epithelial-specific and transformation-sensitive calmodulin-like protein, CLP. We have recently shown that CLP is acting as a light chain for the unconventional myosin-10. Myosin-10 is a highly unstable signaling protein involved in cytoskeletal dynamics, filopodial extension, cell adhesion and migration. We hypothesize that CLP upregulates myosin-10 by increasing its stability, thereby enhancing its function. Specifically, we seek answers to the following questions:

1. Is CLP (and myosin-10) upregulation involved in keratinocyte differentiation?
2. Will cells that over-express CLP/myosin-10 migrate faster to close a wound?
3. How does calmodulin-like protein regulate myosin-10 actin-activated ATPase activity and actin filament motility?
4. What role does Ca2+ play in these processes?

The project uses primary human keratinocytes and involves cell transfections, generation of recombinant proteins in the baculovirus/insect expression system, and the development of functional in vitro and in vivo assays. Studies of myosin-10 function are performed in collaboration with the laboratory of Dr. Tom Burghardt.

Relevance to public health: Loss of CLP is a hallmark of transformed cells and is observed in a majority of breast cancers. Determining the cellular effects of CLP in epithelial cells will advance our understanding of basic Ca2+-mediated processes in epithelial cell growth and differentiation. Identifying the mechanism of myosin-10 regulation by CLP will reveal its role in specific processes linked to changes in cell signaling and cell migration that occur during normal differentiation and upon cellular escape from growth control. This may lead to the development of novel intervention strategies to halt malignant transformation, e.g., through the identification of novel downstream targets of CLP.