Simultaneous Membrane Capacitance Measurements and TIRF Microscopy to Study Granule Trafficking at Immune Synapses

Abstract

Whole-cell capacitance measurements allow the direct measurement of exocytosis with high temporal resolution. An added benefit of the whole-cell configuration is the possibility to control the cytosolic free calcium concentration allowing examination of the role of intracellular calcium in a variety of processes. We have coupled this method with imaging of cytotoxic granule release using total internal reflection fluorescence microscopy (TIRFM) to identify the capacitance steps associated with cytotoxic granule release identified by TIRFM. This requires the use of fluorescent granule markers to identify cytotoxic granules and allows characterization of cytotoxic granule fusion and of the behavior of cytotoxic granules at the immune synapse prior to fusion. Combination of these methods enables the study of a number of processes relevant to the function of the immune synapse.

Three-terminal magnetic tunnel junction synapse circuits showing spike-timing-dependent plasticity

“…The plots are normalized to their maximum values. It can be seen that we closely capture the biological transient behavior with the circuit, which agrees well with simulations, mathematical modeling, and analysis of biological neurons [46][47][48][49][50][51][52][53][54].…”

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses

“…3. None of the individual vesicle pools have been detected in CTLs ( 117 , 170 ). Therefore, presumably no protein is required to halt or maintain LGs in an intermediate release-ready state as is the case in neurons.…”

Alternative UNC13D Promoter Encodes a Functional Munc13-4 Isoform Predominantly Expressed in Lymphocytes and Platelets

“…We next analyzed Munc13-4 isoform trafficking and distribution to the IS. To do this, we took advantage of high-resolution total internal reflection fluorescence (TIRF) microscopy that generates an evanescent wave within ∼200 nm of the plasma membrane allowing a better visualization of vesicle dynamics in the proximity of the IS (18,19,30,31). Freshly isolated CTLs were co-transfected with constructs encoding recombinant Munc13-4-mCherry as well as Granzyme B-TFP and then were placed on coverslips coated with anti-CD3 antibodies to mimic IS formation.…”