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Cell autonomous and non-autonomous control of chromosome segregation.

A major interest of the lab is to undrstand how chromosome segregation is regulated by intracellular and extracellular cues. We study how the physical and geometric properties of a cell control chromosome segregation. We are also exploring the possibility that mitosis is not a cell autonomous process. We investigate how the architecture and organization of a tissue a cell is embedded in impact chromosome segregation.

Size matters: DNA:cytoplasm ratio controls cell division.

Every cell type has its characteristic shape and size. The importance of maintaining correct cellular dimensions for cell division is not understood. We study how cell size affects cell cycle progression in budding yeast and mammals. Our studies in yeast indicate that increasing cell size without increasing genome copy number has a dramatic effect on all aspects of cell proliferation and genome stability (Figure 1).

This finding suggests that maintenance of a cell-type specific DNA to cytoplasm ratio is critical for cell function. Currently, we are investigating which aspects of S phase and mitosis are affected by altering DNA to cytoplasm ratio and how this leads to genomic instability. Whether maintenance of a correct DNA to cytoplasm ratio affects mammalian cell fitness and proliferation is a question that we are also pursuing.

Position matters: Control of exit from mitosis by nuclear division and position.

Correctly positioning the nucleus within the cell is essential for accurate chromosome segregation. If the site of nuclear division is not coordinated with the site of cytokinesis anucleate and multinucleate cells are produced. This coordination is especially critical in budding yeast. In this organism the site of cytokinesis is established long before nuclear division occurs. The nucleus, thus, must be threaded through the bud neck during each anaphase.

In budding yeast control of the Mitotic Exit Network (MEN) is central for ensuring accurate partitioning of the nucleus during mitosis. The Mitotic Exit Network is a Ras-like GTPase signaling cascade that controls the final cell cycle transition, called exit from mitosis. It is known as the Hippo tumor-suppressor pathway in mammals. The Mitotic Exit Network activates the protein phosphatase Cdc14, which triggers mitotic spindle disassembly and cytokinesis.

The localization of Mitotic Exit Network components is essential for coordinating the site of nuclear division with the site of cytokinesis. MEN constituents localize to the cytoplasmic face of spindle pole bodies (SPBs), the budding yeast equivalent of centrosomes (Figure 2).

The MEN activator Lte1 localizes to the bud, the MEN inhibitor Kin4 localizes to the mother cell. The localization patterns of MEN components and MEN regulators lead to a simple hypothesis: The cell is divided into a MEN inhibitory zone in the mother cell, where Kin4 resides, and a MEN activating zone in the bud, where Lte1 resides (Figure 2). The MEN component carrying SPB functions as the sensor. Only when the MEN bearing SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud during anaphase where the MEN activator Lte1 resides can exit from mitosis occur. We showed that spindle position is indeed sensed in this manner. When Lte1 is targeted to the mother cell, cells with mis-positioned spindles inappropriately exit from mitosis. When Kin4 is targeted to the bud, cells with correctly positioned spindles fail to exit from mitosis. Thus, spatial information is sensed and translated into a chemical signal by targeting activators and inhibitors of signal transduction pathways to specific cellular locations. Determining how Kin4 and Lte1 are targeted to the mother cell and bud, respectively, and how these proteins control MEN activity has been a recent focus of our lab.

The Mitotic Exit Network activity is not only controlled in space but also in time - its activity is restricted to anaphase. Central to restricting MEN activity to anaphase is the Polo kinase (Figure 3).

It regulates the activity of the GTPase effector kinase of the MEN, Cdc15. Furthermore, we identified a second regulatory network, known as the FEAR network, that restricts MEN activity to anaphase and it too does so by regulating Cdc15. The FEAR network transiently activates Cdc14 during early anaphase. Cdc14 in turn dephosphorylates multiple MEN components foremost the GTPase effector kinase Cdc15, thereby inducing a “preactivated” state. Once the GTPase Tem1 is then activated as the MEN bearing spindle pole body moves into the bud, full MEN activation occurs.

Together our findings lead to the conclusion that multiple temporal and spatial signals are integrated to control the activity of the Mitotic Exit Network. All these signals converge on Cdc15, the effector kinase of the MEN. These findings have significant implications for GTPase signaling pathway regulation. They indicate that GTPase signaling pathways are not only regulated by the nucleotide binding state of their GTPases but that signal integration occurs at multiple steps in the signaling cascade. We are now studying how MEN activation at the spindle pole body leads to activation of the Cdc14 phosphatase in the nucleolus.

Context matters: Cell non-autonomous control of chromosome segregation by tissue architecture.

Chromosome segregation is considered a cell autonomous process. However, in multicellular organisms, cell division occurs in the context of tissues. Is it possible that tissue architecture affects the chromosome segregation process? We recently begun to study this question. Our findings show that disruption of mammalian epithelial architecture and hence loss of cell shape and polarity decreases chromosome segregation fidelity (Figure 4).

These findings indicate that cell non-autonomous determinants affect chromosome segregation. Understanding the molecular mechanisms underlying this regulation will not only be critical for understanding how chromosome segregation is controlled by tissue context but may also shed light on the origins of genome instability so frequently found in cancer. Loss of tissue architecture is a hallmark of solid tumors and could thus be a cause of chromosome instability in tumor cells.

Recent publications:

Attner MA, Amon A. Control of the mitotic exit network during meiosis. Mol Biol Cell. 2012 Aug;23(16):3122-32.

Chan LY, Amon A. Spindle Position Is Coordinated with Cell-Cycle Progression through Establishment of Mitotic Exit-Activating and -Inhibitory Zones. Mol Cell. 2010 Aug 13; 39(3): 444-454. PMCID: PMC2946186

Falk JE, Chan LY, Amon A. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4. Proc Natl Acad Sci U S A. 2011 Aug 2; 108(31):12584-12590. Epub 2011 Jun 27. PMCID: PMC3150932

Falk JE, Campbell IW, Joyce K, Whalen J, Seshan A, Amon A. LTE1 promotes Exit from Mitosis by multiple mechanisms. Mol Biol Cell. 2016 Oct 26. pii: mbc.E16-08-0563. [Epub ahead of print]

Falk JE, Tsuchiya D, Verdaasdonk J, Lacefield S, Bloom K, Amon A. Spatial signals link exit from mitosis to spindle position. Elife. 2016 May 11;5. pii: e14036. doi: 10.7554/eLife.14036. PMCID: PMC4887205

Rock JM, Amon A. Cdc15 integrates Tem1 GTPase-mediated spatial signals with Polo kinase-mediated temporal cues to activate mitotic exit. Genes Dev. 2011 Sep 15; 25(18): 1943-1954. PMCID: PMC3185966

Rock JM, Lim D, Stach L, Ogrodowicz RW, Keck JM, Jones MH, Wong CC, Yates JR 3rd, Winey M, Smerdon SJ, Yaffe MB, Amon A. Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes. Science. 2013 May 17;340(6134):871-5.