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

Abstract

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.

Fibronectin, Vitronectin, and Collagen I Induce Chemotaxis and Haptotaxis of Human and Rabbit Mesenchymal Stem Cells in a Standardized Transmembrane Assay

“…Taken together, these results raise concerns about the widespread use of BSA-supplemented buffers in cell migration studies. Our observations of reduced migration after longer periods of premigration culture time are also consistent with previous observations where trypsinized fibroblastic cells needed no more then 1 h to form actin-based adhesions (60), and that such complexes, indicative of sessile cellular phenotype (61), are formed with MSCs cultured on polycarbonate membranes (50). Such complexes could explain the relatively long migration time (up to 24 h) needed in these studies (57,58).…”

“…Cells were isolated from the bone marrow of rabbits as reported earlier (50). Femurs from two New Zealand White Rabbits weighing between 3 and 4 kg were used separately.…”

“…role due to the fact that cells that have just migrated have relatively weak adhesions to the substrate (25) and are prone to disruption under suboptimal fixation. We have shown that various nonglutaradehyde fixatives perturb the cytoskeleton and provoke cell detachment from these membranes (50). On the basis of these results, all migration experiments were performed with no premigration attachment time, no BSA added, and glutaradehyde as fixative.…”

Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension

“…Cytoskeletal structures were stained [22] and imaged with a Zeiss LSM 510 confocal microscope. Alexa fluor 546 phalloidin (150 nmol/L) and TOPRO-3 (1 μmol/L) (Invitrogen) were added to stain F-actin and DNA.…”

Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance

“…HeLa, VA13, or MCF7 cells (2.8 ϫ 10 4 cells) were seeded onto eight-well culture slides (BD). The cells were fixed using a protocol based on that described by Thibault and Buschmann (20). Briefly, 1 day after seeding, cells were washed twice with phosphate-buffered saline not containing either calcium ions or magnesium ions [PBS(Ϫ)] and fixed in PBS(Ϫ) containing 1% paraformaldehyde and 0.5% Triton X-100 at 37°C for 20 min.…”