(177x) Dorsoventral Polarity Regulates the Modes and Mechanisms of Cell Migration in Confinement

Authors: 
Mistriotis, P., Johns Hopkins University
Wisniewski, E., Johns Hopkins University
Bera, K., Johns Hopkins University
Law, R., Johns Hopkins University
Tuntithavornwat, S., Johns Hopkins University
Afthinos, A., Johns Hopkins University
Zhao, R., Johns Hopkins University
Kalab, P., Johns Hopkins University
Konstantopoulos, K., Johns Hopkins University
Cell locomotion is a multi-faceted process integral for fundamental biological events, such as organ formation and embryonic development, and also during disease progression including cancer metastasis. Much of what is known about cell motility stems primarily from experiments on two-dimensional (2D), unconfined surfaces which lack key physical and topographical cues. However, in vivo, cells must travel through complex microenvironments including confining 3D pores of varying diameters (1 to 20 µm), or fiber- and channel-like tracks ranging from 3 to 30 µm in width and up to 600 µm in length. These tracks exert physical cues on cells that initiate intracellular signaling cascades which alter cell migration mechanisms.

The nucleus has a rate-limiting role in cell migration through confined spaces. Tumor cell motility is halted at pore sizes smaller than ~7 μm2 due to lack of nuclear translocation, and can only resume following matrix degradation. Nuclear stiffness is considered a key determinant of confined migration as its reduction via lamin-A knockdown enhances migration through narrow pores. Confined migration is commonly studied using microfluidic devices that enable real-time, high-throughput monitoring of cell motility in channel-like tracks of prescribed physical properties. Confinement exerts a mechanical stress on the nucleus, ultimately leading to nuclear envelope rupture and DNA damage with important consequences for its genomic integrity. For cells migrating through openings of the exact same cross-sectional area (Height (H) x Width (W)=constant), the extent of nuclear rupture is more pronounced when the migration tracks have a low ceiling (small height) rather than a narrow base (small width). Cells are compressed vertically (top to bottom) as opposed to laterally (sidewise) in the former and latter cases, respectively. These observations prompted us to address a fundamental and yet unanswered question: are migrating cells endowed with the ability to respond to different geometries of migration tracks by changing for instance the speed, the mode and/or the mechanisms of cell locomotion? We hypothesized that if cells could indeed sense and respond to distinct geometries, this would be due to the intrinsic asymmetry of their molecular machinery and/or cytoskeletal organization along the top-to-bottom cell axis, termed dorsoventral cell polarity. To test this hypothesis, we induced cells with pre-established dorsoventral polarization to migrate inside collagen type I-coated microchannels of a fixed cross-sectional area (30 μm2), which impose either vertical (HxW=3x10 μm2; small height) or lateral (HxW=10x3 μm2; small width) compression on cells.

By integrating microfluidic devices with biophysical tools (Atomic Force Microscopy), imaging techniques (high resolution imaging, FLIM-FRET) and intravital microscopy, we herein demonstrate that dorsoventral polarity directs cell responses in confinement by regulating the modes and mechanisms of cell migration. In vertical confinement, the nucleus poses a mechanical barrier, whose inefficient deformation along the dorsoventral polarity axis mediates bleb-based migration. Lateral confinement, exerted perpendicularly to the dorsoventral polarity axis, activates the perinuclear actin machinery which effectively deforms the nucleus, thereby promoting a faster protrusion-based migration. Thus, key determinants of confined cell locomotion, such as the dynamic interconversion of blebbing versus protrusive modes of migration are influenced by preestablished dorsoventral cell polarization along with nuclear mechanosensing. Our work enhances our understanding of the complex process of confined cell migration and provides a novel perspective on how cells sense and respond to different geometries of migration tracks.