Tracking fluorescent microtubules in living cells

CARLA PALLAVICINI; DIANA E. WETZLER; MARCELO DESPOSITO; VALERIA LEVI; LUCIANA BRUNO

Barcelona

Congreso; The Microtubule Cytoskeleton in Development and Disease; 2013

The cellular cytoskeleton is a dynamic network of polymerized proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. These filaments play an important role in cellular division, structure and intracellular transport. They can exhibit highly nonlinear elasticity and non-thermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing their contribution to complex cellular behaviors, it is necessary to study the dynamics of these filaments in living cells with high spatial precision and temporal resolution. An effective imaging technique to this purpose is confocal fluorescence microscopy; filaments in living cells are marked with fluorescent probes, and movies are obtained. However, crowding, crosswalks between filaments and high background noise levels reduce the efficiency of standard tracking techniques when applied in living cells. In this work we present two tracking algorithms applicable to polymeric structures labeled with fluorescent probes that can be used in cells. The first recovers the coordinates of individual filament segments in consecutive frames with sub pixel resolution. The second algorithm presented is dedicated to tracking filament tips, which can be used, for example, to study microtubules growing processes. In this work, we apply this algorithm to microtubules photobleached according to a pre-defined pattern. This is particularly interesting because it introduces a novel procedure of identifying specific regions of the filaments, without the need of using an external probe. Finally, we present examples of applications of both algorithms to studies of microtubule dynamics in Xenopus Laevis melanophore cells.