Abstract
Cell motility is critical to biological processes from wound healing to cancer metastasis to embryonic development. The involvement of organelles in cell motility is well established, but the role of organelle positional reorganization in cell motility remains poorly understood. Here we present an automated image analysis technique for tracking the shape and motion of Golgi bodies and cell nuclei. We quantify the relationship between nuclear orientation and the orientation of the Golgi body relative to the nucleus before, during, and after exposure of mouse fibroblasts to a controlled change in cell substrate topography, from flat to wrinkles, designed to trigger polarized motility. We find that the cells alter their mean nuclei orientation, in terms of the nuclear major axis, to increasingly align with the wrinkle direction once the wrinkles form on the substrate surface. This change in alignment occurs within 8 hours of completion of the topographical transition. In contrast, the position of the Golgi body relative to the nucleus remains aligned with the pre-programmed wrinkle direction, regardless of whether it has been fully established. These findings indicate that intracellular positioning of the Golgi body precedes nuclear reorientation during mouse fibroblast directed migration on patterned substrates. We further show that both processes are Rho-associated kinase (ROCK) mediated as they are abolished by pharmacologic ROCK inhibition whereas mouse fibroblast motility is unaffected. The automated image analysis technique introduced could be broadly employed in the study of polarization and other cellular processes in diverse cell types and micro-environments. In addition, having found that the nuclei Golgi vector may be a more sensitive indicator of substrate features than the nuclei orientation, we anticipate the nuclei Golgi vector to be a useful metric for researchers studying the dynamics of cell polarity in response to different micro-environments.
Highlights
The organization and reorganization of intracellular structures and organelles is key to the complex biological processes of both cell motility and collective cell behaviors at the tissue scale
Cell motility is directed along wrinkles in active and static wrinkled substrates As we have previously observed [9], nuclear cell tracking by ACTIVE showed that cells on non-wrinkled substrates have no preference in how they explore space (Fig 3D–3F), while cells on both active (Fig 3G–3I) and wrinkled (Fig 3A–3C) substrates showed a strong preference to travel along the direction of forming and established wrinkle direction
We quantified the relationship between nuclei orientation and the orientation of the Golgi body relative to the nucleus before, during, and after exposure to a change in shape memory polymer (SMP) substrate topography designed to trigger polarized motility, and our findings suggest that the displacement vector between the nucleus and Golgi body (NGV) may be a more sensitive indicator of substrate features than the nuclei orientation
Summary
The organization and reorganization of intracellular structures and organelles is key to the complex biological processes of both cell motility and collective cell behaviors at the tissue scale. The repositioning of the Golgi apparatus contributes to polarized cell migration by facilitating the efficient transfer of Golgi-derived vesicles, via microtubules, to the cells leading edge [5, 6]. These vesicles provide the membrane and associated proteins necessary for directed lamellipodial protrusion [7]. The timing of Golgi repositioning in relation to changes in overall cell morphology and intracellular signaling remain poorly understood
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