DS0405 -

How telomeres control cell proliferation potential: a single cell lineage approach – InTelo

Submission summary

This proposal aims at determining the mechanisms involved in telomere-dependent replicative senescence. We expect to understand how telomere shortening, cryptic telomere damage, and signaling at telomeres control entry into and escape from senescence.

In animals, aging tissues accumulate senescent cells, a process considered as tumor-suppressor in youth, but which underlies the loss of regeneration capacity of organs and is ultimately detrimental to the organism late in life. Telomerase inactivation is a major cause of replicative senescence in human somatic cells. Telomerase elongates telomeres and in its absence, telomeres shorten because the semi-conservative DNA replication machinery incompletely replicates DNA extremities. As telomeres shorten, it is thought that their function in preventing chromosome ends from being recognised as accidental chromosomal breaks and subsequently repaired by the DNA damage response (DDR) pathway, is impaired. Thus, in cells lacking telomerase, telomeres reach a critical short length, activate the DNA damage checkpoint, and induce replicative senescence. However, progress in understanding telomere replication, DDR signalling and its relationship to senescence has been hindered by intrinsic variations in telomeres and the stochastic nature of senescence onset.

In this proposal, we plan not only to overcome this barrier, but mainly, to tackle the cause of cell-to-cell variations in the establishment of the onset of senescence. We will do this by combining unique tools we developed to assess replicative senescence in telomerase-negative cells of the model organism Saccharomyces cerevisiae. These tools allow to (1) manipulate and trace single telomeres in single cells; (2) image consecutive cell divisions with high spatial and temporal resolutions from telomerase inactivation to cell death using a microfluidic device; and (3) generate testable mathematical models that will serve to build a comprehensive model of replicative senescence. Using this strategy, we recently discovered that telomerase inactivation in Saccharomyces cerevisiae not only leads to gradual telomere shortening and a permanent cell cycle arrest, but also to recurrent early cell cycle delays (EDs) in some long-lived cell lineages. Cells with this phenotype persist only at low frequency in bulk cultures, making them undetectable in conventional population-averaged assays. Based on data obtained in several mutant backgrounds, we propose a working model where telomere replication defaults, left unrepaired in the absence of telomerase, cause the EDs. Cell cycle can then resume after DNA damage checkpoints extinction. This unexpected phenotype would thus provide an explanation for the time-dependent emergence of checkpoint deregulations and genomic instability in telomerase-negative cells, hallmarks of aging and cancer transformation.

Here, we will build on these observations to dissect the mechanisms involved and test our model. Our multidisciplinary experimental strategies will be based on the one hand in the introduction of mutations in telomerase-negative context and inspecting their effects on the dynamics of senescence onset using the microfluidics system. On the other hand, we will develop fluorescent markers to track in the individual cell lineages identified telomeres, the status of the DDR, genome stability and several cellular organelles. Such high-precision analyses will fuel a global mathematical model of replicative senescence. This project should lead to a profound advance in our understanding of telomere biology and the control of the DDR pathway, and to the discovery of new links between telomeres and other cell components. Our project will potentially foster the development of novel therapies to age-related diseases, cell regeneration impairment and cancer.

Project coordination

Maria Teresa TEIXEIRA (Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LBMCE Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes
IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire
Inria de Paris Institut National de Recherche en Informatique et Automatique

Help of the ANR 431,572 euros
Beginning and duration of the scientific project: February 2017 - 48 Months

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