Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events

The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.