Analyzing Angiotensin II Receptor Type 1 Clustering in PC12 Cells in Response to Hypoxia Using Direct Stochastic Optical Reconstruction Microscopy (dSTORM).

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (AT1R) is expressed in acute O2 sensitive cells, including carotid body (CB) type I cells and pheochromocytoma 12 (PC12) cells, and Ang II increases cell activity. While a functional role for Ang II and AT1Rs in increasing the activity of O2 sensitive cells has been established, the nanoscale distribution of AT1Rs has not. Furthermore, it is not known how exposure to hypoxia may alter the single-molecule arrangement and clustering of AT1Rs. In this study, the AT1R nanoscale distribution under control normoxic conditions in PC12 cells was determined using direct stochastic optical reconstruction microscopy (dSTORM). AT1Rs were arranged in distinct clusters with measurable parameters. Across the entire cell surface there averaged approximately 3 AT1R clusters/µm2 of cell membrane. Cluster area varied in size ranging from 1.1 × 10-4 to 3.9 × 10-2 µm2. Twenty-four hours of exposure to hypoxia (1% O2) altered clustering of AT1Rs, with notable increases in the maximum cluster area, suggestive of an increase in supercluster formation. These observations could aid in understanding mechanisms underlying augmented Ang II sensitivity in O2 sensitive cells in response to sustained hypoxia.

Conformational States Control Lck Switching between Free and Confined Diffusion Modes in T Cells.

T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

How does T cell receptor clustering impact on signal transduction?

The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.