Monitoring Chk1 kinase activity dynamics in live single cell imaging assays.

The ATR/Chk1 pathway is an important regulator of cell cycle progression, notably upon genotoxic stress where it can detect a large variety of DNA alterations and induce a transient cell cycle arrest that promotes DNA repair. In addition to its role in DNA damage response (DDR), Chk1 is also active during a non-perturbed S phase and contributes to prevent a premature entry into mitosis with an incompletely replicated genome, meaning the ATR/Chk1 pathway is an integral part of the cell cycle machinery that preserves genome integrity during cell growth. We recently developed a FRET-based Chk1 kinase activity reporter to directly monitor and quantify the kinetics of Chk1 activation in live single cell imaging assays with unprecedented sensitivity and time resolution.

Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome.

In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor.

AURKA destruction is decoupled from its activity at mitotic exit but is essential to suppress interphase activity.

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation and allosteric interaction with TPX2. Activity peaks at mitosis, before AURKA is degraded during and after mitotic exit in a process strictly dependent on the APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1) and a novel FRET-based AURKA biosensor to investigate how AURKA activity is regulated in the absence of destruction.

Re-investigating PLK1 inhibitors as antimitotic agents.

Polo-like kinase 1 (PLK1) plays key roles during mitosis, prompting the development of PLK1 inhibitors for anticancer therapy. We recently determined that PLK1 is crucially required for entry into mitosis. Hence, we discuss the potential and limitations of PLK1 inhibition strategies to promote mitotic arrest and death of cancer cells.

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1.

Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression.

Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1.

Deciphering the spatio-temporal regulation of entry and progression through mitosis.

Mitosis has been studied since the early 1880s as a key event of the cell division cycle where remarkable changes in cellular architecture take place and ultimately lead to an equal segregation of duplicated chromosomes into two daughter cells. A detailed description of the complex and highly ordered cellular events taking place is now available. Many regulators involved in key steps including entry into mitosis, nuclear envelope breakdown, microtubule (MT) spindle formation, and chromosome attachment, as well as mitotic exit and cytokinesis, have also been identified.

Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis.

The CyclinB1-Cdk1 kinase is the catalytic activity at the heart of mitosis-promoting factor (MPF), yet fundamental questions concerning its role in mitosis remained unresolved. It is not known when and how rapidly CyclinB1-Cdk1 is activated in mammalian cells, nor how its activation coordinates the substantial changes in the cell at mitosis. Here, we have developed a FRET biosensor specific for CyclinB1-Cdk1 that enables us to assay its activity with very high temporal precision in living human cells.

Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.

The cyclin B-Cdk1 kinase triggers mitosis in most eukaryotes. In animal cells, cyclin B shuttles between the nucleus and cytoplasm in interphase before rapidly accumulating in the nucleus at prophase, which promotes disassembly of the nuclear lamina and nuclear envelope breakdown (NEBD). What triggers the nuclear accumulation of cyclin B1 is presently unclear, although the prevailing view is that the Plk1 kinase inhibits its nuclear export.

Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis.

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis.

Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells.

Centriole assembly plays an important role in centrosome duplication during the cell cycle and is a prerequisite for cilia formation during the differentiation of ciliated cells. In spite of numerous investigations, the molecular machinery that governs centriole/basal body formation remains enigmatic. Recent reports suggest that the ubiquitously expressed mammalian centrins, centrin2p and centrin3p, could be involved in the centriole duplication process.

Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons.

Stathmin is a ubiquitous cytosolic phosphoprotein, preferentially expressed in the nervous system, and the generic element of a protein family that includes the neural-specific proteins SCG10, SCLIP, and RB3 and its splice variants, RB3' and RB3". All phosphoproteins of the family share with stathmin its tubulin binding and microtubule (MT)-destabilizing activities. To understand better the specific roles of these proteins in neuronal cells, we performed a comparative study of their expression, regulation, and intracellular distribution in embryonic cortical neurons in culture.

Drosophila stathmin: a microtubule-destabilizing factor involved in nervous system formation.

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes.