Conserved C-terminal domains of mCenp-F (LEK1) regulate subcellular localization and mitotic checkpoint delay.

Centromeric Protein-F (Cenp-F) family members have been identified in organisms from yeast to human. Cenp-F proteins are a component of kinetochores during mitosis, bind to the Rb family of tumor suppressors, and have regulatory effects on the cell cycle and differentiation; however, their role in these processes has not been resolved. Here, we provide evidence that the role of murine Cenp-F (mCenp-F, also known as LEK1) remains largely conserved and that the domains within the C-terminus collectively function to regulate the G2/M cell cycle checkpoint. Overexpression of the C-terminal domain of mCenp-F decreases DNA synthesis. Analyses of deletion mutants of mCenp-F reveal that the complete C-terminal domain is required to delay cell cycle progression at G2/M. Signal transduction pathway profiling experiments indicate that the mCenp-F-mediated cell cycle delay does not involve transcriptional activity of key cell cycle regulators such as Rb, E2F, p53, or Myc. However, endogenous mCenp-F colocalizes with pRb and p107, which demonstrates in vivo protein-protein interaction during cell division. These observations suggest that the domains of the C-terminus of mCenp-F have a conserved function in control of mitotic progression through protein-protein interaction with pocket proteins, thus providing a direct connection between cell cycle regulation and mitotic progression.

Western array analysis of cell cycle protein changes during the hyperplastic to hypertrophic transition in heart development.

Cardiac myocytes proliferate most rapidly during the hyperplastic phase of heart development; however, the level of cell cycle activity is drastically down regulated after birth. Further growth of the heart is achieved by hypertrophic growth of cardiac myocytes. The mechanism that controls the switch from hyperplastic proliferation to hypertrophic growth in cardiac myocytes is unknown. Understanding this fundamental mechanism of cardiac myocyte biology would be most beneficial for studies directed towards myocardial regeneration. In this study, we identified changes in the expression of proteins involved in cell cycle regulation during the hyperplastic to hypertrophic transition of cardiac myocytes. Using a high-throughput immunoblotting technique, we examined 200+ proteins in primary cultures of cardiac myocytes at different developmental time points to determine the important regulators of this transition. In addition, we also analyzed samples from an immortalized cardiac myocyte cell line to compare expression levels of cell cycle regulatory proteins to our primary cultures. Our findings by this uncovered proteomic screen identified several potential key regulatory proteins and provide insight into the important components of cardiac myocyte cell cycle regulation.

Novel 3D culture system for study of cardiac myocyte development.

Insufficient myocardial repair after pathological processes contributes to cardiovascular disease, which is a major health concern. Understanding the molecular mechanisms that regulate the proliferation and differentiation of cardiac myocytes will aid in designing therapies for myocardial repair. Models are needed to delineate these molecular mechanisms. Here we report the development of a model system that recapitulates many aspects of cardiac myocyte differentiation that occur during early cardiac development. A key component of this model is a novel three-dimensional tubular scaffold engineered from aligned type I collagen strands. In this model embryonic ventricular myocytes undergo a transition from a hyperplastic to a quiescent phenotype, display significant myofibrillogenesis, and form critical cell-cell connections. In addition, embryonic cardiac myocytes grown on the tubular substrate have an aligned phenotype that closely resembles in vivo neonatal ventricular myocytes. We propose that embryonic cardiac myocytes grown on the tube substrate develop into neonatal cardiac myocytes via normal in vivo mechanisms. This model will aid in the elucidation of the molecular mechanisms that regulate cardiac myocyte proliferation and differentiation, which will provide important insights into myocardial development.