Role of Weightlessness on Metabolism (Actin) - 12.03.13
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Utrecht University, Utrecht, , Netherlands
European Space Agency (ESA)Sponsoring Organization
Information PendingResearch Benefits
Information PendingISS Expedition Duration:
October 2003 - October 2004Expeditions Assigned
8,9Previous ISS Missions
This experiment aims at studying the effect of weightlessness on the structure and metabolism of cellular actin microfilaments in mammalian cells. Actin is one of two proteins involved in muscle contraction and is found in both smooth and striated muscle. It also serves as an important structural molecule.
Information PendingEarth Applications
Information PendingOperational Protocols
The experiment makes use of an established cell line i.e. mouse fibroblasts C3H 10T1/2, originating from the American Type Culture Collection. All experiments are performed at 370C in plunger box units according to the protocols described below. The plunger box units (PBUs) consist of two culture compartments. PBUs have the left hand compartment labelled compartment A and the right hand compartment labelled compartment B. All culture compartments contain C3H10T1/2 cells plated on a cover slip and medium. Each culture compartment has 3 fluid chambers leading into the compartment: from left to right, chamber 1, chamber 2, and chamber 3. Chamber 1 is filled with medium and activated prior to flight. Chamber 2 is filled with Platelet-Derived Growth Factor (PDGF) in medium and activated in flight. Chamber 3 is filled with a fixing lysis solution and is activated in flight. These chambers can be activated by their respective plungers, denoted plunger 1, plunger 2, and plunger 3. These numbers are preceded by an A or B to indicate which culture compartment they are associated with. The aim of this experiment is to establish the effect of weightlessness on the actin microfilament system of C3H 10T1/2 cells, activated or not activated with growth factor, using (immuno) fluorescence microscopy. After the containers arrive at the ISS at ambient temperature (15-250C), the containers are incubated at 370 C for 6 hours. After this incubation period plungers 2A and 2B (medium and PDGF) are activated for all 8 containers. Plunger 3A and 3B (fixing lysis buffer) are activated at 4 different time points, 0 minutes (actin 1), 5 minutes (actin 2), 10 minutes (actin 3), and 30 minutes (actin 4).
The observed effects of microgravity on cell morphology suggested that the actin microfilament system was sensitive to gravity conditions. Therefore, some preliminary experiments were performed during two sounding rocket flights in which the cells were treated or not with EGF according to standard protocols. After fixation and recovery of the cells, the actin filament system was labeled specifically with fluorescent-labeled phalloidin, a toxin known for its high affinity for filamentous actin. Analysis of the cells by fluorescence microscopy and by fluorometry according standard procedures revealed clearly that the F-actin content of the cells was increased under microgravity conditions. Addition of EGF caused a further increase in the amount of F-actin, but no difference in activation was observed under normal and microgravity conditions. These preliminary observations, however, clearly indicate that the actin microfilament system may represent a gravity-sensitive cell component.