Author: Fatimah Al-Rahbi, Layla Al-Saadi, Yasir Al-Mansoori
Research Article
Control of T Lymphocyte Morphology by the GTPase Rho
Layla Al-Saadi¹*, Yasir Al-Mansoori² and Fatimah Al-Rahbi³
¹Department of Microbiology, Sultan Qaboos University, Muscat, Oman
²Department of Biotechnology, Dhofar University, Salalah, Oman
³Department of Virology, University of Nizwa, Nizwa, Oman
Published: 22 February 2019
Abstract
Background: T lymphocytes undergo dynamic morphological changes crucial for their functions, including migration through tissues, formation of immune synapses, and effector responses. These shape changes depend on the reorganization of the actin cytoskeleton. Rho GTPases are key regulators of actin dynamics and actomyosin contractility. While roles for Rac and Cdc42 in T cell activation and migration are established, the specific contribution of RhoA to controlling T cell morphology remains less defined.
Objective: This study aimed to investigate the role of the RhoA GTPase, and its downstream effector ROCK, in regulating the morphology and actin cytoskeleton organization of T lymphocytes.
Methods: Primary human T lymphocytes were used. RhoA activity was measured using Rhotekin-RBD pull-down assays following stimulation with chemokines (SDF-1α) or TCR engagement (anti-CD3/CD28). The effects of inhibiting RhoA activity (using C3 exoenzyme) or ROCK activity (using Y-27632) on T cell morphology were assessed using phase-contrast microscopy, quantitative shape analysis (circularity, aspect ratio), and fluorescence microscopy after F-actin staining with phalloidin. The specific impact on cell polarity, including leading-edge formation and uropod structure, was examined in migrating T cells on ICAM-1 coated surfaces. Live-cell imaging was used to monitor morphological dynamics.
Results: RhoA activity was rapidly and transiently increased in T lymphocytes upon stimulation with SDF-1α or anti-CD3/CD28 antibodies. Inhibition of RhoA using C3 exoenzyme or inhibition of ROCK using Y-27632 resulted in significant morphological changes. Treated T cells often adopted a more rounded phenotype and exhibited impaired polarization, particularly a reduction or loss of the distinct trailing uropod structure typically seen in migrating or activated lymphocytes. Phalloidin staining revealed that Rho/ROCK inhibition decreased the accumulation of F-actin and likely actomyosin contractility in the cell cortex and uropod region. Live-cell imaging confirmed that Rho/ROCK inhibition impaired the retraction of the cell rear during chemokine-induced migration.
Conclusion: These findings demonstrate that the RhoA GTPase plays a critical role in controlling T lymphocyte morphology. By activating its downstream effector ROCK and regulating actomyosin contractility, RhoA is essential for establishing and maintaining cell polarity, particularly the formation and retraction of the uropod. This function is likely crucial for efficient T cell migration and potentially other processes requiring defined cell shape and cortical tension, such as immune synapse stability.
Keywords: T Lymphocyte; Rho GTPase; RhoA; ROCK; Cell Morphology; Actin Cytoskeleton; Actomyosin Contractility; Uropod; Cell Polarity; Cell Migration.
