Optical manipulation of the cell cytoskeleton

The use of shorter pulsed lasers for intracellular nanosurgery is of growing interest to understand complex biological processes. Here, we develop a femtosecond laser ablation system capable of irradiating multiple fluorescently labeled cytoskeletal elements. Transmission electron microscopy analysis of femtosecond irradiated regions of the cell demonstrate the laser's ability to target submicron regions of the cell. Ultrastructural analysis suggests no significant advantages over lasers with the shorter pulse durations.;Irradiation of single microtubules demonstrates the effectiveness of the setup to precisely target a 0.25 micron structure, without affecting surrounding microtubules or regions within the cell. The efficiency of this irradiation process further facilitates the study of dynamics for microtubule depolyermization. Analysis of depolymerization rates confirms a statistically significant dependence on the time after irradiation as well as the relative location within the cell.;The femtosecond laser system was then applied to the irradiation of the centrosome in migrating cells. The fluorescing centrosome region was targeted in multiple cell models including wound healing kidney epithelial cells (PtK2), single migrating human osteosarcoma cells (U2OS), and single fast migrating human neutrophils (HL60). Irradiation of the centrosome in U2OS cells resulted in a loss of polarized cell morphology at 30 to 90 minutes following laser exposure. This loss of polarization was associated with significant changes in the microtubule and actin network. Irradiation of the centrosome in wound edge PtK2 cells resulted in similar effects. Targeted PtK2 cells lost their ability to directionally migrate and often fell behind the neighboring control migrating cells. Ablation of the centrosome in neutrophils before and after chemoattractant was added resulted in a loss of polarized cell morphology. Unstable lamellae-like protrusions were often formed at random positions around the cell, however none were sufficient for migration in any direction.;Responses of the various cell types to centrosome irradiation, in combination to the selective targeting of microtubules surrounding (but not including) the centrosome suggest a novel role of the centrosome. These studies suggest the centrosome maintains cell polarization through signal mediation rather than mechanically through the microtubule network.