Question

Immunology

How does Indo1 allow measurement of signaling in real time? What is store operated calcium entry and how is it regulated?

If the EF-hand domain was mutated so it behaved like it was always bound to calcium how would this impact signaling?

Give 2 examples of inhibitory receptors that regulate T cell function How does CTLA4 reduce signaling in T cells?

Describe 3 ways that Fas/FasL interactions regulate T cell mediated immune responses

Answer 1

Calcium is an important second messenger and Indo-1 is a ​​​Calcium- sensitive fluorophore with the excitation wavelength in the UV range. So When excited, the emitted fluorescence is measured which can be used in the detection of Ca2+ levels in real time.

Store-operated calcium entry is a ubiquitous Ca2+ entry pathway that is activated upon the stimulation of plasma membrane receptors which are coupled to PIP2 hydrolysis and IP3 generationbin response to depletion of internal Ca2+ stores of endoplasmic reticulum. It is a highly regulated process where STIM molecules act as Ca2+ sensors within the ER and when Ca2+ store depletion happens, these rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels .

The EF hand motiff is responsible for binding of intracellular calcium.Binding of calcium to this domain leads to a conformational change from “closed” to “open,” exposing a hydrophobic surface that binds to a target protein/ peptide. If there happens a mutation in the EF- hand such that it always remains bound to Ca2+ all the signalling process including enzyme activity, permeability of ion channels, activity of ion pumps and components of cytoskeleton would be affected because Ca2+ is an important secondary messenger. This will lead to disregulated cell function and ultimately apoptosis.

Examples of inhibitory receptors that regulate T- cell function include PD-1, BTLA, CTLA-4, Lag-3, CD200:CD200R and some Ly49 family members.

CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits an inhibitory signal to T cells. However the complete mechanism is unknown. It is believed that CTLA-4 recruits a phosphatase to the T cell receptor which leads to termination of the signal and hence reduced signalling. Recent work has suggested that CTLA-4 may function by capturing and removing B7-1 and B7-2 from the membranes of antigen-presenting cells, thus making these unavailable for triggering of CD28 hence reducing the stimulatory signal.