Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse

Abstract

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis towards the IS.

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

“…Therefore, PI values were normalized by cell size and shape (Fig. 4) (25) (86) and measured the relative ability of MTOC and MVB to polarize towards the IS.…”

T Lymphocyte and CAR-T Cell-Derived Extracellular Vesicles and Their Applications in Cancer Therapy

“…This supports that an altered actin reorganization at the IS may cause the defective MVB orientation occurring in PKCδ-interfered Th lymphocytes [ 31 ]. More recently, PKCδ-dependent depolymerization of the F-actin pool surrounding the MTOC/MVB ( Figure 3 ) has been shown to be involved in MTOC/MVB polarization to the Th IS [ 32 , 33 , 69 ]. It is well known that a microtubule network is required for the transport of late endosomes, since MTOC co-migrates with diverse secretory granules (cytolytic granules/MVB in CTL, cytokine secretory granules/MVB in Th lymphocytes) [ 13 , 32 , 120 ].…”

“…In addition, externalization of phosphatidylethanolamine (PE) and phosphatidylserine (PS), that binds Annexin V, occurs in plasma membrane-derived EV and, to a lower extent, in exosomes. Bold, underlined characters identify those molecular components or processes that regulate MVB secretory traffic in T lymphocytes: lysosomal trafficking regulator (LYST) [ 14 ], neutral sphingomyelinase 2 (nSMase2) [ 15 ], DAG [ 16 ], diacylglycerol kinase α (DGKα) [ 17 , 18 , 19 , 20 ], acidic sphingomyelinase (aSMase) [ 21 ], MAL [ 22 , 23 ], ISGylation [ 24 ], Adaptor protein 3 (AP3) [ 25 ], Rab27a [ 26 ], Rab11, Rab7 [ 27 ], dynein [ 28 ], kinesin-1 [ 29 ], cortical F-actin [ 30 , 31 ], centrosomal area F-actin [ 32 , 33 ], protein kinase C δ (PKCδ) [ 31 , 34 ], protein kinase C θ (PKCθ) [ 35 , 36 ], vesicle-associated membrane protein 8 (VAMP-8) [ 37 ], syntaxin 4 (STX4) [ 38 ], syntaxin 7 (STX7) [ 39 ], syntaxin 8 (STX8) [ 40 ], syntaxin 11 (STX11) [ 41 ], SNAP23 [ 38 ]. Underlined characters identify molecules involved in shedding vesicles generation in T lymphocytes: tumor susceptibility gene 101 (TSG101) and vacuolar protein sorting 4 (VPS4) [ 42 ].…”