Image

Research > Team Biology of Quiescent Cells

Biology of Quiescent Cells

Principal Investigator : Isabelle Sagot

sagot-top

Team Members

Laetitia Gouleme - ASI UB

Damien Laporte - CRCN CNRS

Isabelle Sagot - DR2 CNRS

 

Summary

Cells perpetually face the decision to proliferate or to enter a non-dividing state. Quiescence, a state defined as a reversible arrest of proliferation, is the most common cellular situation found on earth, as it concerns all kind of cells, from microbes to human stem cells. Quiescent cells not only have to survive and face age but they must also preserve their ability to re-enter the cell cycle in a tightly regulated manner, and give rise to a healthy progeny. Therefore quiescence is at the heart of crucial biological issues including development, aging and evolution. Yet, astonishingly little is known about the molecular determinants that orchestrate quiescence establishment and exit.

We are using both single cell eukaryotes and mammalian models to study the cell biology of quiescence. In the past years, we have shown that upon quiescence entry, cells assemble specific structures that display original properties, including actin and microtubule hyper-stable arrangements. Our project is to study these structures and use them as tools to address central questions such as: how do quiescent cells survive and what are the cascade of molecular switches that tightly control the transitions between quiescence and proliferation.

 

Research Activity

Quiescent cells are confronted with quite a few challenges. On the one hand, quiescent cells need to preserve their ability to proliferate, sometimes over several years. As they age, quiescent cells must cope with extrinsic or intrinsic harmful events that cause the accumulation of damaged macromolecules. The inability to handle these stresses can ultimately lead to cell death. Additionally, for the sake of tissues, organisms and the species itself, quiescent cells must produce offspring born “damage-free”. On the other hand, cells must enter quiescence and return into the proliferation cycle in a tightly regulated manner, uncontrolled transitions being potentially deleterious for the whole organism, as exemplified by stem cell depletion or cancer. Finally, in the case of micro-organisms competing for their environmental niche, quiescence exit must be swift to ensure the prevalence of the species. Therefore quiescence is at the heart of crucial biological issues including development, aging and evolution.

Few years ago, we have pioneered the characterization of quiescent yeast cells at the cellular level. We have shown that upon entry into quiescence two evolutionary distant yeast species, S. cerevisiae and S. pombe, re-organize drastically some of their intracellular machineries. Indeed, upon carbon starvation cells assemble two specific structures Actin Bodies (AB) and Proteasome Storage Granules (PSGs) that respectively result from the specific reorganization the actin cytoskeleton and the proteasome (Sagot et al, 2006; Sahin et al, 2008; Laporte et al 2008). Importantly, these structures are mobilized within seconds when cells exit quiescence upon re-feeding.

 

sagot-fig1
In red the Actin cytoskeleton – left : in dividing S. cerevisiae (Actin patches and cables) ; right : in quiescent S. cerevisiae (Actin Bodies). In green the proteasome - left : in the nucleus of dividing S. cerevisiae; right : in quiescent S. cerevisiae in proteasome storage granules.

Using AB and PSGs as new specific markers of the quiescent state at the single cell level, we have demonstrated that quiescence entry and exit can occur not only in the G1 phase of the cell cycle, but also in other cell cycle stages. Additionally, we have established that the cell’s metabolic status rather than cell cycle regulators is critical for the control of quiescence/proliferation transitions (Laporte et al 2001).

 

sagot-fig2
Quiescent cells in all cell cycle stages. Left : Nucleus (blue – DAPI) and Spindle Pole Body (green). Right : Cell wall (ConA-FITC – Green) and Actin Bodies (Alexa-Phalloidin – Red).

More recently, we have shown that the microtubule cytoskeleton is drastically rearranged upon entry into quiescence. In proliferating S. cerevisiae, microtubules are highly dynamic structures assembled by the spindle pole body (SPB, the yeast equivalent of the centrosome). As the SPB is embedded into the nuclear membrane, two microtubule populations can be distinguished: the cytoplasmic microtubules, that position the nucleus, and the nuclear microtubules that are required for chromosome segregation during mitosis. In the G1 phase of the cell cycle, each chromosome centromere is attached via its kinetochore complex to a short nuclear microtubule (<300nm) and while centromeres are highly clustered next to the SPB, most of the telomeres are localized into 8 to 10 foci distributed over the nuclear periphery. The nucleolus, built onto the rDNA, is opposed to the SPB on the other half of the nucleus. This organization is reminiscent of the so-called Rabl configuration that has been described in fly, salamander and plants and strictly depends on nuclear microtubules. We have shown that upon quiescence entry, while cytoplasmic microtubules vanish, S. cerevisiae cells assemble a long and stable monopolar array of nuclear microtubules that spans the entire nucleus. Consequently, the nucleolus is displaced, and, as kinetochores remain attached to microtubule tips, microtubule elongation and stabilization cause the massive rearrangement of chromosomes. Finally, when cells exit quiescence, the nuclear microtubule array slowly depolymerises, and, by pulling attached centromeres back to the SPB, allows the recovery of a typical Rabl-like configuration Therefore, in S. cerevisiae, upon quiescence entry, microtubules are stabilized and cause the massive rearrangement of the nucleus. Importantly, we have found that mutants that are not capable of stabilizing a nuclear microtubule array are incapable to survive in quiescence, suggesting that this reorganization is required for facing age. (Laporte et al, 2013 ; Laporte and Sagot, 2014).

 

sagot-fig3
Quiescent S. cerevisiae displaying a microtubulue bundle (green) inside the nucleus (nuclear envelope in red). The cell wall is stained with calcofluor white revealing the bud scars (blue).

We are now deciphering the molecular steps required for the formation of these quiescent cells specific structures and trying to identify their physiological functions. We are also scrutinizing actin and microtubule reorganization in quiescent S. pombe and in mammalians cells.

 

Key and active collaborations

 

Fundings 2009-14

 

Keywords

Quiescence - Actin - Microtubule - Proteasome - S. cerevisiae - S. pombe - Live cell imaging

 

Selected publications 2009-2018

Laporte D, Jimenez L, Gouleme L, Sagot I. Yeast quiescence exit swiftness is influenced by cell volume and chronological age. Microb Cell. 2017 Dec 6;5(2):104-111

Laporte D, Courtout F, Tollis S, Sagot I. Quiescent Saccharomyces cerevisiae forms telomere hyperclusters at the nuclear membrane vicinity through a multifaceted mechanism involving Esc1, the Sir complex, and chromatin condensation. Mol Biol Cell. 2016 Jun 15;27(12):1875-84

Laporte D, Courtout F, Pinson B, Dompierre J, Salin B, Brocard L, Sagot I. A stable microtubule array drives fission yeast polarity reestablishment upon quiescence exit. J Cell Biol. 2015 Jul 6;210(1):99-113

Laporte D, Sagot I. Microtubules move the nucleus to quiescence. Nucleus. 2014 Mar-Apr;5(2):113-8

Jimenez L, Laporte D, Duvezin-Caubet S, Courtout F, Sagot I. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation. J Cell Sci. 2014 Feb 15;127(Pt 4):719-26

Laporte D, Courtout F, Salin B, Ceschin J, Sagot I. An array of nuclear microtubules reorganizes the budding yeast nucleus during quiescence. J Cell Biol. 2013 Nov 25;203(4):585-94

Daignan-Fornier B, Sagot I. Proliferation/Quiescence: When to start? Where to stop? What to stock? Cell Div. 2011 Dec 9;6(1):20

Daignan-Fornier B, Sagot I. Proliferation/quiescence: the controversial "aller-retour". Cell Div. 2011 May 9;6:10

Laporte D, Lebaudy A, Sahin A, Pinson B, Ceschin J, Daignan-Fornier B, Sagot I. Metabolic status rather than cell cycle signals control quiescence entry and exit. J Cell Biol. 2011 Mar 21;192(6):949-57