T lymphocytes are components of the adaptive immune response that circulate throughout the body in search of antigen presenting cells bearing peptide-MHC complex molecules. T cells are inherently polarized with an anterior leading edge and a posterior protrusion called the uropod. While several details of T cell polarity have been established, the source and significance of this morphology is still not completely understood. Septins are components of the cytoskeleton important for structural integrity and cell cycle control. They form heteromeric complexes that interact with both actin filaments and microtubules and have diverse biological functions. Therefore, we decided to investigate the role of septins in T cell morphology.
Septin complexes are found at the cell cortex and are enriched at the T cell mid-body. In order to assess the functional requirements of septins in T cells, we designed short hairpin RNAs (shRNAs) targeting Septins 1, 6, 7 and 9. Targeting of Sept7 resulted in the reduction of the entire septin complex, a result that was only observed when Sept7 was targeted. T cells lacking septin complexes have a pronounced increase in uropod length. To determine a potential mechanism for this result, we examined the activation state of myosin II in septin deficient T cells. In these cells, we did not observe any differences in the phosphorylation of either the myosin light chain or heavy chain, although myosin II activity was still required for uropod formation.
Although septin deficient T cells crawled at normal velocities, they exhibited structural instabilities including membrane blebbing and the presence of excess protrusions. Septin deficient T cells also had a slight reduction in actin filaments. Therefore, it is not clear if the membrane defects we observed during crawling are from the reduction in actin filaments, reduction in the septin cytoskeleton or a combination of both. Finally, loss of septins did not affect functions that require cell polarization; however, septin deficient T cells rapidly permeated extremely small pores, consistent with the loss of a structural scaffold. Thus, septins play a critical role in T cell morphology and structural integrity and may be important for transendothelial migration.