Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

PSL Research University, Institut Curie

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.

The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis.

In spite of conspicuous differences in their polarized architecture, swimming unicellular eukaryotes and migrating cells from metazoa display a conserved hierarchical interlocking of the main cellular compartments, in which the microtubule network has a dominant role. A microtubule array can organize the distribution of endomembranes owing to a cell-wide and polarized extension around a unique nucleus-associated structure. The nucleus-associated structure in animal cells contains a highly conserved organelle, the centriole or basal body. This organelle has a defined polarity that can be transmitted to the cell. Its conservative mode of duplication seems to be a core mechanism for the transmission of polarities through cell division.

Organelle positioning and cell polarity.

Polo-like kinase 4 (PLK4) is a unique member of the Polo-like family of kinases that shares little homology with its siblings and has an essential role in centriole duplication. The turn-over of this kinase must be strictly controlled to prevent centriole amplification. This is achieved, in part, by an autoregulatory mechanism, whereby PLK4 autophosphorylates residues in a PEST sequence located carboxy-terminal to its catalytic domain. Phosphorylated PLK4 is subsequently recognized by the SCF complex, ubiquitinylated and targeted to the proteasome for degradation. Recent data have also shown that active PLK4 is restricted to the centrosome, a mechanism that could serve to prevent aberrant centriole assembly elsewhere in the cell. While significant advances have been made in understanding how PLK4 is regulated it is certain that additional regulatory mechanisms exist to safeguard the fidelity of centriole duplication. Here, we overview past and present data discussing the regulation and functions of PLK4.

Polo-like kinase 4: the odd one out of the family.

The microtubule-based flagellum, or basal body/axoneme, is ancestral: it is a so-called "derived character" defining the extant eukaryote branch, present at the apparition of the early eukaryotic cell. It is a genuine cell compartment. Its structural conservation throughout evolution is remarkable, as well as the molecular conservation of the intraflagellar transport genes. From the start, it links cell sensation and cell locomotion, two properties which require integrated wiring at the cell level. The cell division machinery has been also tightly coupled with this organelle throughout evolution, possibly to ensure the continuity of this cellular polarized organization through division. The association of three basic cell modules, locomotion, sensation and division, all expressing a facet of cell polarity, within the same apparatus, could have given optimal survival value to primitive eukaryotic cells and could explain the evolutionary conservation and success of the basal body/axoneme. In vertebrates, primary cilia are critical sensory organelles, including during early mammalian development. Today the eukaryotic flagellum is not only an important issue for basic cell biology and developmental biology but also for human pathology. Many apparently unrelated severe developmental pathologies turned out to be all connected to the assembly or the function of the primary or beating cilia. The current efforts on human ciliopathies benefit directly from long lasting basic research on the flagellar assembly in experimental models like Chlamydomonas reinhardtii.

Michel Bornens

CNRS,
UMR 144,
Molecular Mechanisms of Intracellular Transport,
CNRS, UMR 144, Molecular Mechanisms of Intracellular Transport

PSL Research University, Institut Curie

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Michel Bornens

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PSL Research University, Institut Curie

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

CNRS,
UMR 144,
Molecular Mechanisms of Intracellular Transport,
CNRS, UMR 144, Molecular Mechanisms of Intracellular Transport