(G-256) Confinement drives memory via mitochondrial localization and vinculin-containing focal adhesion formation

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row G - Poster # 256

Co-Author(s)

JM

Jenna A. Mosier

Graduate Student
Vanderbilt University, United States

Presenting Author(s)

Emily D. Fabiano

Graduate Student
Vanderbilt University
Nashville, Tennessee, United States

Co-Author(s)

CL

Catherine Ludolph

Undergraduate Student
Vanderbilt University, United States
AW

Addison White

Undergraduate Student
Vanderbilt University, United States

Primary Investigator(s)

CR

Cynthia Reinhart-King, PhD (she/her/hers)

Professor; BMES, President
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN
Nashville, Tennessee, United States

Introduction:: Breast cancer metastasis is still a poorly understood process [1]. The primary tumor microenvironment requires cells to first migrate through dense tissue organized into confining pores [2-3]. Migration through confinement significantly increases cell migration and energy utilization [4-5]. Here, we use a collagen microtrack platform to mimic tumor confinement in vitro to investigate the effects of confinement on breast cancer cell migration memory.

Materials and Methods:: MDA-MB-231 breast cancer cells were seeded into confined (i, 7 µm), unconfined (ii, 15 µm), and temporarily confined microtracks (iii, 7 µm region then 15 µm region) (Fig. 1A) and monitored using phase or confocal microscopy and velocity was tracked by manually outlining cells in Fiji. TMRM was used to monitor mitochondrial positions during migration. CRISPR/ Cas9 was used to generate vinculin-knockout cells (VclKO) to assess the effects of vinculin depletion on cell migration.

Results, Conclusions, and Discussions::

Results: Confinement increases both cell velocity (Fig. 1B) and front:rear (F:R) active mitochondrial distribution (Fig. 1C). Additionally, priming cells in confinement results in maintenance of high migration velocity (Fig. 1D) and F:R mitochondria (Fig. 1E) upon exit from confined spaces, up to 200 µm past transitioning from the confined to unconfined region. Disrupting focal adhesions (FAs) in VclKOs, as shown by imaging for the FA protein paxillin (Fig. 1F), significantly decreases F:R active mitochondrial localization in confinement (Fig. 1G). Further, in temporarily confined microtracks, VclKOs do not maintain their exit velocity after priming in confinement and transitioning to an unconfined region (Fig. 1H), suggesting that vinculin recruitment and mitochondrial localization are integral for priming.

Discussion: It has previously been shown that proper FA formation is required for persistent migration [6], as well as intact mitochondrial function and localization in the cell [7]. Here, disrupting FAs by knocking-out vinculin results in the loss of mitochondrial F:R localization and memory of previous confinement. Our data suggest that the loss of vinculin reduces directionality of migrating cells and prevents proper mitochondrial polarization to the cell front, disrupting the ability of cells to maintain high speeds after exit.

Conclusions: Spatial confinement leads to increased velocity and leading-edge mitochondria in breast cancer cells, fueling mechanical memory of previous confinement that may aid in navigating the tumor microenvironment. Furthermore, disrupting mitochondrial localization through focal-adhesion disruption results in the loss of priming ability. Further understanding of the link between mitochondrial dynamics and cell migration is essential for developing key therapeutics for metastasis.

Acknowledgements (Optional): :

References (Optional): : [1] Lyons&Traina, AEMB. 2019. [2] Irimia&Toner. Integr. Biol. 2009. [3] Pathak&Kumar, PNAS. 2012. [4] Zanotelli, et al. Nat Comm, 2019. [5] Mosier*, Rahman-Zaman*, et al. Biophys J. 2019. [6] Rahman-Zaman, et al. Mol Biol Cell. 2016. [7] Kawada, et al. Canc Bio. 2013.