Centrosome positioning in polarized cells: common themes and variations.

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.

Adhesive micropatterns to study intermediate filament function in nuclear positioning.

The nucleus is generally found near the cell center; however its position can vary in response to extracellular or intracellular signals, leading to a polarized intracellular organization. Nuclear movement is mediated by the cytoskeleton and its associated motors. While the role of actin and microtubule cytoskeletons in nuclear positioning has been assessed in various systems, the contribution of intermediate filaments is less established due in part to the lack of tools to study intermediate filament functions. The methods described here use micropatterned substrates to impose reproducible cell shape and nucleus position. Intermediate filament organization can be perturbed using gene downregulation or upregulation; intermediate filaments can also be visualized using fluorescent intermediate filament proteins. This protocol is valuable for characterizing the role of intermediate filaments in a variety of live or fixed adherent cells.