Mitochondria tether to Focal Adhesions during cell migration and regulate their size.

Mitochondria are the key generators of ATP in a cell. Visually, they are highly dynamic organelles that undergo cellular fission and fusion events in response to changing cellular energy requirements. Mitochondria are now emerging as regulators of mammalian cell motility. Here we show that mitochondria infiltrate the leading edge of NIH3T3 fibroblasts during migration. At the leading edge, we find that mitochondria move to and tether to Focal Adhesions (FA). FA regulate cell migration by coupling the cytoskeleton to the Extracellular Matrix through integrin receptors. Importantly, we find that inhibition of mitochondrial ATP generation concomitantly inhibits FA size. This suggests that mitochondrial energy production regulates migration through FA control. 3X with PBS. Cells were imaged on the Zeiss AxioObserver Z1 microscope using a 63X, 1.4 NA objective and deconvolved using Zen blue edition under fast iterative parameters and bad pixel correction. Spatial reconstruction of images were generated using surface rendering on Imaris software. The quantitation of focal adhesions immunostained for vinculin was performed using ImageJ.

In addition, mitochondria contribute to Ca 2+ homeostasis, and the metabolism of amino acids, lipid and nucleotides [4,5]. Dysfunction of mitochondrial metabolism and dynamics contributes to cancer development [6] and a broad spectrum of human diseases [7]. Mitochondria are highly dynamic organelles with fission and fusion events continually reshaping their morphology [8].
Mitochondria vary in size from individual organelles in the submicron length range to large interconnected tubular networks spanning the cytoplasm. It is now recognized that contact between mitochondria and other organelles is an important part of cellular physiology and homeostasis [9,10]. For example, contact with the Endoplasmic Reticulum licenses mitochondrial fission [11,12] and mitochondrial derived membranes are part of the peroxisome biogenesis pathway [13].
Mitochondria are emerging as novel regulators of cell motility [14,15]. Cell migration regulates several important physiological processes, among them embryonic development, tissue morphogenesis and the immune response [16]. Dysregulated migration is often associated with cancer development and metastatic progression [17]. Fragmented mitochondria are found to infiltrate the leading edge of breast and ovarian cancer cell during migration [14,15]. Inhibition of either mitochondrial ATP generation [14] or Ca 2+ uptake through silencing of the Mitochondrial Calcium Uniporter (MCU) [15] inhibits motility.
An unresolved question, however, is to what part of the cell migration machinery do mitochondria regulate and what motility structures do they move and tether to. In this report, we find that during fibroblast migration mitochondria move to and tether to Focal Adhesions (FA).
FA are multi-protein adhesive structures on the basal cell surface that couple the extracellular All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/827998 doi: bioRxiv preprint matrix (ECM) to intracellular actin fibers via clusters of transmembrane integrin receptors [18,19]. FA allow for the propagation of mechanical forces within the cell and into the external environment [18,19]. We find that inhibition of mitochondrial ATP generation decreases FA size, identifying FA as a migratory structure regulated by mitochondrial contact and ATP generation.
All rights reserved. No reuse allowed without permission.

Results & Discussion
Mitochondria move to the leading edge during migration. In both breast and ovarian cancer cells, mitochondria infiltrate into the leading edge lamellipodia [14,15,20]. To determine whether or not this was the case in non-transformed cells, we used live cell microscopy to image mitochondria in freely migrating NIH3T3 mouse fibroblasts (Fig 1a, Supplementary Video 1). In these cells, we observe that as the cell moves forward, multiple mitochondria infiltrate the leading edge. To extend these findings, we tracked individual mitochondria in NIH3T3 cells expressing a fluorescently tagged Cortactin protein (Fig 1b, Supplementary Video 2). Cortactin localises prominently to the lamellipodial edge in migrating cells and regulates cell migration by binding to the Arp 2/3 complex, stabilizing actin branches and activating multiple signaling pathways necessary for motility [21]. As shown in Fig 1b, multiple mitochondria with various starting locations move towards the Cortactin polarized edge (red arrow) during migration (direction indicated by white arrow). In migratory NIH3T3 cells, as in other cell types [22,23], mitochondria move along microtubules from the cell interior (Fig 1c) . However, once in the leading edge, which is thinner than the majority of the cell body, they do not associate with tubulin (Fig 1c). Thus, the presence of mitochondria at the leading edge does not absolutely require microtubule dependent processes.
Mitochondria contact focal adhesions during migration. The infiltration of mitochondria towards the migratory leading edge suggests an important role for them in sustaining cell migration.
Previous reports indicate that regulation of calcium flux via the Mitochondrial Calcium Uniporter (MCU) and energy levels via the AMP-activated protein kinase (AMPK) are two pathways through which mitochondria affect migration [14,15]. However, it is unknown what migratory structures in lamellipodia mitochondria might be interacting with.
All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/827998 doi: bioRxiv preprint We speculated that mitochondria might be interacting with Focal Adhesions (FA) since these adhesive structures position themselves at the periphery of a cell and are frequently found at or close to the lamellipodial edge. To test this idea, we used live cell microscopy to simultaneously visualize mitochondria and the Talin protein during fibroblast migration (Fig 2A and Supplementary Video 3). Talin is a component of FA that links transmembrane integrin receptors to actin fibers in the cytosol [24]. We reasoned that mitochondria might regulate cell migration by interacting with FA since functioning FAs are required for many forms of mammalian cell motility [25]. Inhibition of mitochondrial activity reduces Focal Adhesion size. We next examined what functional role mitochondria might have on FA structure. First, we used the TMRE dye to measure mitochondrial membrane potential in migrating cells. As shown in Fig 3A, mitochondria tethered to FA have varied mitochondrial membrane potential, some exhibiting high membrane potential while others did not. To determine whether or not mitochondrial activity had any relationship with FA structure, we treated NIH3T3 cells with oligomycin. Oligomycin inhibits mitochondrial ATP generation by preventing ATP synthase activity [26] and has been previously been shown to retard migration of ovarian cancer cells [14]. We treated cells with oligomycin and used an image analysis program [27] to quantitate FA number and size four hours later. As shown in Fig 3B, the All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Interaction between mitochondria and other cellular structures is now understood to be an important aspect of the homeostasis of multiple organelles [9]. Mitochondria make important functional contacts with the endoplasmic reticulum (ER), vacuoles and peroxisomes [9,13]. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/827998 doi: bioRxiv preprint Mitochondrial contact with the ER regulates mitochondrial division [12] and also allows ERderived lipids to move to mitochondria. With respect to non-membrane bound structures, mitochondria are dependent on microtubules for their transport and Kinesin and Dynein motor proteins link mitochondria to tubulin. Our work shows that Focal Adhesions are among the list of organelles and cellular structures interacting with mitochondria and is consistent with the idea that mitochondria regulate cell migration, at least in part, by regulating cellular adhesion. Plus 488 (A32723, Thermofisher). Cells were then washed 3X with PBS. Cells were imaged on the Zeiss AxioObserver Z1 microscope using a 63X, 1.4 NA objective and deconvolved using Zen blue edition under fast iterative parameters and bad pixel correction. Spatial reconstruction of images were generated using surface rendering on Imaris software. The quantitation of focal adhesions immunostained for vinculin was performed using ImageJ. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.   The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.  2014) Step-by-step quantitative analysis of focal adhesions. MethodsX 1: 56-9 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.