P06 | MICROGRAVITY-INDUCED CHANGES IN CYTOSKELETON STRUCTURE OF TCAM-2 CELLS

M. Berardini¹, L. Gesualdi¹, A. Di Pauli¹, M. Signore², S. Broccolucci¹, F. Ferranti³, M. Bizzarri⁴, M. Tafani⁴, G. Ricci⁵, A. Catizone¹ | ¹Dept of Anatomy, Histology, Forensic-Medicine and Orthopedics,“Sapienza” University of Rome, Rome, Italy; ²RPPA Unit, Proteomics Area, Core Facilities, Istituto Superiore di Sanità, Rome, Italy; ³Human Spaceflight and Scientific Research Unit, Italian Space Agency, Rome, Italy; ⁴Dept of Experimental Medicine “Sapienza” University of Rome, Rome, Italy; ⁵Dept of Experimental Medicine, Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Italy

Cytoskeleton, a mechano-sensing apparatus of the cells, is sensitive to gravity conditions¹. In our previous study, we investigated the effects of simulated microgravity (SM) on the cytoskeleton of human male germ cells (TCam-2). We observed that SM for 24 h altered microtubule and actin organization². To determine whether cytoskeletal changes occur during the early stages of SM exposure, we analysed the effects after a short 3 h period. Quantitative analyses were performed using Reverse Phase Protein Array (RPPA). The distribution pattern of cytoskeletal filaments was studied by indirect immunofluorescence (IIF) and Confocal microscopy, using maximum projections obtained from 1μm optical sections. RPPA analyses showed an increase in total cofilin and tubulin as early as 3h of exposure. Cellular cofilin exists in two forms: cofilin and phospho-cofilin (the latter represents the inactive form), both regulating actin polymerization and dynamics in the cytoplasm. However, both forms are also present in the nucleus, where they transiently influence gene regulation in response to specific stimuli. This dual localization highlights the dynamic regulatory role of cofilin phosphorylation in cellular processes³. Indirect IIF showed that, in SM samples, total cofilin levels increased while nuclear phospho-cofilin levels decreased. Consistent with RPPA data, tubulin levels also increased according to IIF. These observations suggest a possible perturbation of actin cytoskeleton caused by SM exposure, which in turn also affects tubulin polymerization and organization confirming the already known interaction between microtubules and microfilaments⁴. Future studies are necessary to better understand the biological significance of these modifications. Nonetheless, these results indicate that cytoskeleton remodelling occurs in TCam-2 cells as early as 3 h after SM exposure, providing further insight into how TCam-2 cells adapt to SM condition.