Adaptation and Changes in Actin Dynamics and Cell Motility as Early Responses of Cultured Mammalian Cells to Altered Gravitational Vector

Abstract

Cultured mammalian cells have been shown to respond to microgravity (μG), but the molecular mechanism is still unknown. The study we report here is focused on molecular and cellular events that occur within a short period of time, which may be related to gravity sensing by cells. Our assumption is that the gravity-sensing mechanism is activated as soon as cells are exposed to any new gravitational environment. To study the molecular events, we exposed cells to simulated μG (SμG) for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h using a three-dimensional clinostat and made cell lysates, which were then analyzed by reverse phase protein arrays (RPPAs) using a panel of 453 different antibodies. By comparing the RPPA data from cells cultured at 1G with those of cells under SμG, we identified a total of 35 proteomic changes in the SμG samples and found that 20 of these changes took place, mostly transiently, within 30 min. In the 4 h and 8 h samples, there were only two RPPA changes, suggesting that the physiology of these cells is practically indistinguishable from that of cells cultured at 1G. Among the proteins involved in the early proteomic changes were those that regulate cell motility and cytoskeletal organization. To see whether changes in gravitational environment indeed activate cell motility, we flipped the culture dish upside down (directional change in gravity vector) and studied cell migration and actin cytoskeletal organization. We found that compared with cells grown right-side up, upside-down cells transiently lost stress fibers and rapidly developed lamellipodia, which was supported by increased activity of Ras-related C3 botulinum toxin substrate 1 (Rac1). The upside-down cells also increased their migratory activity. It is possible that these early molecular and cellular events play roles in gravity sensing by mammalian cells. Our study also indicated that these early responses are transient, suggesting that cells appear to adapt physiologically to a new gravitational environment.

Photoactivatable surfaces resolve the impact of gravity vector on collective cell migratory characteristics

“…Therefore, remodeling of cytoskeletal systems induced by the cell inversion against the gravity vector is necessary for safe nuclear translocation. Contrary to a previous report, another report observed cytoskeletal remodeling within a faster time frame (10 min) for single cells; this report revealed a transient reduction in actin stress fiber formation with a considerable increase in lamellipodia formation, followed by the complete loss of such changes within one or two hours [ 6 ]. Therefore, the cellular response to gravity is time-dependent; the transient effect shows lower actin stress fibers, whereas the adaptive effect shows higher actin stress fibers.…”

“…However, there might be a difference between the intrinsic migration velocity for single cells compared to cells in a group, especially when there are significant alterations in the biological functions of cells in groups compared to single cells, such as cellular adhesion, migration persistence [ 18 ], and drug sensitivity [ 19 , 20 ]. In contrast, Ju et al recently reported that upon inversion against gravity, single cells showed enhanced motility [ 6 ], which could be due to the intrinsic difference between the various cell lines or the difference between the substrates used for cell culture. Next, we examined whether the proliferation ratio was affected by the gravity vector, and the proliferation ratio was calculated by counting the number of cells just before the cluster expansion (0 h) and at 14 h after expansion.…”

“…Thus, free actin moieties would be utilized in lamellipodia extension and the appearance of leader cells, in a similar trend to the upright position; however, surprisingly, it showed a higher emergence of leader cells in the inverted positions than in the upright positions. A previous study discussed the enhanced activity of Arp2/3 upon inverting single cells against the gravity vector [ 6 ], even though it will not contribute to actin network branching and cell protrusions due to poor actin resources in the vicinity of the strong peripheral actomyosin bundles [ 33 ]. It is expected that the blebbistatin treatment released the actin molecules from the bundles and makes it available for actin polymerization and lamellipodia extension together with highly active Arp2/3, thereby stronger lamellipodial extension and leader cell formation were observed in the inverted position.…”

“…Another critical biological function affected by gravity is cell migration. Bovine aortic endothelial cells (BAECs) exhibit upregulated motility when they are sparsely cultured and inverted against gravity, whereas confluent cells exhibit delayed motility when the wound scratch assay is performed [ 6 ]. These discrepancies in migratory behavior can be attributed not only to the collective or single-cell motility characteristics but also to the essential methodological limitations of the conventional wound healing assays [ 7 ].…”

Fluid and Bubble Flow Detach Adherent Cancer Cells to Form Spheroids on a Random Positioning Machine

“…The human follicular thyroid carcinoma FTC-133 cell line (passages 8-18) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The MCF-7 mammary carcinoma cells (passages 5-9) and PC-3 prostate carcinoma cells (passages [36][37][38][39][40] were purchased from ATCC (Manassas, VA, USA). The Calu-3 lung carcinoma cell line (passage 19) was a kind gift from Prof. Heike Walles, the University of Magdeburg.…”