RhoGTPases as key players in mammalian cell adaptation to microgravity

Abstract

A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions.

Figure 1

Central role of the RhoGTPases in the integrated response of mammalian cell to microgravity-related conditions. A growing number of studies are revealing that cells reorganize their cytoskeleton, modulate intracellular tension, and initiate nuclear shapes changes when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches. RhoGTPases are known for cytoskeletal dynamics control; nevertheless they are also involved in many other aspects as discussed in this review. We identify general principles dependent on RhoGTPases and define cell sensitivity to gravitational stresses such as oxidative stress, intracellular tension, cell-cell and cell-ECM adhesions, and Wnt/β-catenin pathways. We will try to establish that integrated cellular responses in microgravity are related to specific RhoGTPase activities.

Figure 2

RhoGTPase actions on the cytoskeleton and cell dynamics (modified from [14]). Integrins are necessary for translating the mechanical properties of the extracellular environment into intracellular RhoGTPase-signaling pathways. RhoA influences filopodia formation and focal adhesion assembly and maturation, in addition to controlling stress fiber formation and intracellular tension. Rac1 primarily controls actin assembly and formation of lamellipodia to ensure cell migration. Fibrillogenesis is controlled positively by RhoA and negatively by Rac1. Both RhoA and Rac1 are controlled by specific activators (GEF) and inhibitors (GAP, GDI). Cell adaptation to mechanical/gravitational challenges triggers activation of pathways integrated by RhoGTPases.

Figure 3

Role of AMPc on RhoGTPases activities and commitment of multipotent cells. Microgravity affects the growth, proliferation, and differentiation of multipotent cells by increasing AMPc production. AMPc contributes to cytoskeleton reorganization as it regulates negatively RhoA activity. Limitation of osteoblastogenesis might be linked to the ability of microgravity to reduce RhoA and Rac1 activities. RhoA and Rac1 activations support osteoblasts differentiation for their respective role in ERK activation and beta-catenin nuclear translocation. Sustained adipogenesis observed in microgravity-related condition might be linked to ability of AMPc to trigger integrin a5b1/a6b1 switch. Signaling through a6b1 integrin is known to support adipogenesis. A direct activation of adipogenic transcription factors (cEBPs) by AMPc has been also described.

Figure 4

RhoA and Rac1 activities are downregulated after 6 days of culture in simulated-microgravity conditions. Cultures were performed with C3H10T1/2 (multipotent embryonic cells) on collagen-coated microbeads (Cytodex 3, Sigma) for adipogenic induction and on Cytodex 3 beads coated with apatite minerals complexed to collagen for an osteogenic one. The adipogenic media contained 1 μM of rosiglitazone and the osteogenic media 5 mg/mL of L-ascorbic acid, β-glycerophosphate at 10⁻³ M, and retinoic acid at 10⁻⁵ M, inαMEM. Microbeads with cells were cultured for 2 days in 90 mm petri dishes (untreated for culture) with 10 mL of proliferation media (αMEM), after which the cells were switched 2 days in differentiated media, and finally left for 6 days in a NASA rotating wall vessel (RWV). In parallel, controls were realized by culturing beads in petri dishes. RhoA and Rac1 active assays were performed with specific G-LISA kits (cytoskeleton). The positive controls were pure active proteins of RhoA and Rac1 provided with the kit. The results are expressed as percentage of the positive controls; they show standard error of the mean (SEM) of samples extracted from three independent experiments and are compared with Student's statistical t-test.

Figure 5

Proposed models describing the regulations of RhoA and Rac1 activities in space-related conditions. On Earth MSCs are well spread and exhibit a tensed cytoskeleton in particular of microtubules, intermediate filaments, and actin stress fibers associated with stable focal adhesions within the extracellular matrix. These elements are controlled by GTPases RhoA and Rac1. We hypothesize that during short-term exposure to microgravity, RhoA might be inhibited to allow cytoskeleton reorganization in respect to the new mechanical status. Cell tension reduction might be mandatory during this adaptation. At the same time, Rac1 is activated to control peripheral actin polymerization and induces ROS production. All these events lead rapidly to a rounder cell shape with disorganization of microtubules, stress fibers, intermediate filaments, and focal adhesions. Transcription may be also altered as nucleus shape is changed. In these conditions, cell is still able to migrate. When exposure to microgravity is prolonged both RhoA and Rac1 might be inhibited explaining decreases in osteogenesis and myogenesis and enhancement of adipogenesis of MSCs. In addition, RhoA inhibition limits fibrillogenesis (a tension-dependent process); extracellular matrix is not properly synthesized and lost its mechanical properties appearing softer for MSCs, reinforcing adipogenesis. At that time, migration is inhibited, consistent with cytoskeleton alterations and Rac1 decrease. MSCs become very round, have low adhesion, and may initiate anoikis.