Tony Ly

Lab members

Van Kelly

Cell state transitions during cell growth and division

Our research aim is to characterise the proteomic changes that accompany and control cell state transitions during cell growth and division in human cells. To achieve this aim, we use a combination of state of the art techniques, including fluorescence-activated cell sorting (FACS) and quantitative mass spectrometry (MS)-based proteomics.

We will focus initially on cell state transitions that occur during the mitotic cell division cycle. This cycle can be separated into four major phases (G1, S, G2, and M) that are largely defined by two major processes: DNA replication (S phase) and chromosome segregation (Mitosis). Cells can also enter a reversible quiescent state, called G0.

Mitosis can be further resolved into discrete subphases based on changes in cellular architecture that can be visualized by light microscopy, or by immunostaining for specific molecular signaling events, including phosphorylation of histone H3 and degradation of cyclin proteins (e.g. cyclin A and cyclin B). Unlike in mitosis, it is less clear if gap phases are similarly structured as a linear sequence of state transitions. Evidence in support of a linear progression model during gap phases is the proposed existence of an ‘antephase’ during G2. Antephase is a short time window late in G2 that precedes nuclear envelope breakdown and chromatin condensation that is characterized by an increased sensitivity towards DNA damage. On the other hand, recent studies suggest gap phases of the cell cycle are characterized by bifurcations in cell fate trajectories, leading to heterogeneous, temporally aligned cell states.

Comprehensive, molecular definitions of cell state and identity can be obtained using quantitative mass spectrometry-based proteomics (Ly et al. eLife 2014). Recent developments in mass spectrometry (MS) enable the high throughput identification and quantitation of thousands of proteins in a single analysis. Multidimensional analysis of the proteome is now possible. Static and dynamic parameters of proteins can be measured, including protein copy numbers, post translational modifications, protein-protein interactions, and protein half-life.

We developed a method combining FACS and MS to measure protein changes during mitosis proteome-wide in an asynchronous culture of human leukemia cells (Fig. 1A).

Using this method, we aim to dissect cell state transitions in the mitotic cell division cycle using quantitative, multidimensional proteomics as comprehensive readouts of cellular state (Fig. 1B).

Selected publications:

*Ly, T., Whigham, A., Clarke, R., Brenes-Murillo, A.J., Estes, B., Madhessian, D., Lundberg, E., Wadsworth, P., *Lamond, A.I. (2017). Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS. eLife 6, e27574. *Co-corresponding authors

Ly, T., Endo, A., Lamond, A.I. (2015). Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells. eLife 4, e04534.
 
Ly, T., Ahmad, Y., Shlien, A., Soroka, D., Mills, A., Emanuele, M.J., Stratton, M.R., Lamond, A.I. (2014). A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells. eLife 3, e01630.