Actin Depletion Initiates Events Leading to Granule Secretion at the Immunological Synapse

Abstract

SummaryCytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

“…Upon engagement by anti-CD3, we observed a rapid reduction in cortical actin density at the center of the synapse, giving rise to a ring-like actin structure (Fig. 1C, Upper Far Left), as previously reported (6,10). This reduction in actin density was followed by the movement of lytic granules into the TIRF field ( Fig.…”

“…To evaluate the relationship between actin dynamics and lytic granule secretion more fully, we applied a TIRF-based imaging approach to T-cell activation on a planar surface, permitting a rapid, high-resolution view of the T-cell synapse throughout stimulation; this approach was suitable for imaging granule fusion at the plasma membrane (21). To follow the fate of lytic granules, CTLs were cotransfected with Lifeact-mApple and LAMP1-EGFP, a marker for lytic granules (10). Cells were activated on anti-CD3-coated glass, allowing us to visualize cortical actin density and lytic granule secretion simultaneously over time.…”

“…Acute reduction of cortical actin density has been shown to regulate the access of secretory granules to the plasma membrane and to facilitate secretion in a number of cell types (7,8). Notably, CTL recognition of a target is accompanied by rapid changes in cortical actin, resulting in a dramatic reduction of actin density at the interface between the CTL and its target (9,10). Moreover, this reduction in cortical actin in CTLs is spatially and temporally correlated with the secretion of lytic granules (10).…”

“…Notably, CTL recognition of a target is accompanied by rapid changes in cortical actin, resulting in a dramatic reduction of actin density at the interface between the CTL and its target (9,10). Moreover, this reduction in cortical actin in CTLs is spatially and temporally correlated with the secretion of lytic granules (10). Conversely, elevated cortical actin density at the immunological synapse has been correlated with defective lytic granule secretion (11,12).…”

Cytoskeletal control of B cell responses to antigens

“…To understand how signalling pathways interact with cytoskeletal structures to achieve specific mechanical and regulatory functions, future studies will likely benefit from improvements in these areas. Advances in optical microscopy have been making long strides towards three-dimensional high-resolution imaging and it will be exciting to see this new technology employed for studies of immune cells 76, [163][164][165] . Genetic manipulation is more accessible and precise than ever before, and new optogenetic and nanomechanical methods offer unprecedented temporal and spatial control of manipulation of live cells 166,167 .…”