Molecular mechanisms underlying the coordination of cell growth and cell division – Growth and Division

The mechanisms that estblish a polarity axis during the cell cycle are highly dynamic, involving signaling proteins such as Cdc42 and its regulators that are present in very low amounts in the cell. The methods employed to monitor these proteins must therefore be sufficiently sensitive.

We are using interdisciplinary approaches including high spatial and temporal resolution live cell imaging to monitor vesicle dynamics during the cell cycle. Moreover, we are developing new quantitative tools to understand how vesicle dynamics change during the cell cycle-dependent establishment of a polarity axis. These approaches are complimented by mathematical modelling. Modelling is important, since the behaviour of complex signaling systems is often not intuitive and modelling provides a means of predicting the behaviour of a system that would not have been evident from experiments alone. To test the predictions of such modelling, we use the genetically tractable model eukaryote budding yeast. Budding yeast are also amenable to biochemistry and we have been testing how phosphorylation of Cdc42 regulators by cyclin dependent kinase 1 (Cdk1) results in the establishment of a polarity axis.

The high resolution imaging system that we have built has enabled us to carry out a quantitative imaging-based screen to identify mutants that disrupt the cell-cycle specific organization of membrane trafficking domains. This work is ongoing and we will submit a manuscript on this project in late 2012/early 2013.

The imaging system has enabled us to identify a role for Cyclin Dependent Kinase 1 (Cdk1) in the control of polarized growth that extends beyond Cdk1's known role in regulating actin dynamics (See Publication 1 and 3 below).

We have also used the high spatial and temporal resolution of the system to track the movement of intracellular vesicles in live cells and identify the presence of a Rho GTPase Activating Protein (Rho GAP) on these vesicles. These results provide a mechanism that explains the spatial regulation of Rho GTPase activation, a feature of Rho GTPase biology that is presently poorly understood (See publication 2 below).

We have also used our imaging system with complimentary quantitative approaches and mathematical modelling to understand how a polarity axis is established in a cell cycle-specific fashion. Our exciting new results have uncovered a new mechanism involved in the cell cycle-dependent establishment of a polarity axis involving endocytosis. This work is submitted for publication and received encouraging reviews to which we are currently responding (See publication 4 below).

Our imaging system and quantitative image approaches are facilitating the analysis of dynamic processes including cell growth and cell cycle progression in live cells. The combined use of this system with powerful mathematical modelling and genetic approaches has enabled us to identify new mechanisms and players involved in specifying the axis of cell growth and division. A major question that emerges is whether these newly identified mechanisms are conserved in other eukaryotic cells. The fact that the proteins that we have identified are highly conserved, and that some have been implicated in human diseases including cancer suggest that our results may be relevant and that such proteins may represent good candidates for therapeutic intervention.

Publications:
1) Cdk1-dependent control of membrane trafficking dynamics.
Kellogg D*, Royou A, Velours C, McCusker D*. Mol Biol Cell. 2012 Jul 5. [Epub ahead of print]. * Equal contribution.
2) Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.
Lefèbvre F, Prouzet-Mauléon V, Hugues M, Crouzet M, Vieillemard A, McCusker D, Thoraval D, Doignon F. Eukaryot Cell. 2012 May;11(5):590-600. Epub 2012 Mar 23.
3) Coordinating membrane addition and cell cycle progression.
McCusker D. and Kellogg D. Curr. Opinion Cell Biol. Invited submission. In preparation.
4) Robust polarity establishment via an endocytosis-based cortical corralling mechanism.
Jose M, Tollis S, Nair D, Sibarita, JB and McCusker D. Journal of Cell Biology. Submitted.

Molecular mechanisms that coordinate cell growth and cell division
The dynamic properties of the cytoskeleton facilitate cell growth and cell division. The pattern and amount of cell growth, combined with the timing of cell division, generates the spectrum of cell sizes and shapes found in nature(1). Despite the fundamental importance of these processes, little is known about how cell growth and cell division are coordinated(2). In a manuscript that we recently submitted, an anonymous reviewer commented that, « Understanding the mechanisms that coordinate cell growth and cell division represents one of the key remaining frontiers in Cell Cycle Biology. »

Some 30 years ago, it was recognized that a core component of the yeast cell division machinery, Cdk1, is required to initiate polarization of the actin cytoskeleton and morphogenesis of a daughter cell in late G1 of the cell cycle(3). Cdk1 associated with G1 cyclins promotes polarized growth, however, the mechanisms used by Cdk1 to trigger these events are poorly understood(4). Budding yeast is an excellent model organism to study the cell cycle dependent trigger that initiates polarized growth because the proteins controlling the cell cycle and cell polarity are highly conserved from yeast to mammals(5, 6). During my postdoctoral work, I identified substrates of G1 cyclin-Cdk1 that trigger cell polarization in yeast and I made the surprising discovery that Cdk1 activity is also required for the maintenance of cell surface growth (7) (reviewed in(8-11)). Specifically, I found that:
(1) Cdk1 is required for polarized cell surface growth during a normal cell cycle.
(2) A major target of G1 cyclin-Cdk1 activity is a novel multi-protein complex that I purified and identified by mass spectrometry comprising the GEF, GAP, scaffold and adaptors of the Cdc42 GTPase module.
(3) Mapping of phosphorylation by mass spectrometry and analysis of phosphorylation site mutants revealed that G1 cyclin-Cdk1 regulation of the module is both extensive and critical for polarized cell surface growth.

Exploring the mechanisms by which Cdc42 is activated by Cdk1 will therefore improve our understanding of how cell growth and cell division are coordinated, a key unresolved issue in cell biology. Specifically, the projects outlined in my research proposal will provide significant insights into:
(1) The molecular mechanisms by which Cdk1 activates Cdc42 to initiate and maintain polarized growth.

(2) Mechanisms that contribute to endocytic and exocytic vesicle segregation to establish cell polarization.