# Using optogenetics to dissect the role of signaling complex formation and organization in T-Cell activation

> **NIH NIH F31** · PRINCETON UNIVERSITY · 2020 · $28,650

## Abstract

PROJECT SUMMARY
 CAR-T therapy relies on the creation of synthetic T-cell receptors (TCR) to recognize cancer
cells antigens as well as activate the full immune response, something endogenous TCRs cannot do on
their own. However, this reliance on synthetic receptor molecules limits both the range and
effectiveness of cancer immunotherapy. Developing new modes of cancer immunotherapy depends
on finding new targets that activate the full T cell signaling response. One such target may be the
signaling complex that forms immediately proximal to the TCR between the scaffold protein LAT and
its kinase ZAP70. This signaling complex appears to organize all the downstream pathways activated
following antigen binding. Moreover, it was recently found in vitro that LAT and ZAP70 form liquid
droplets in a process termed “protein phase separation” and that these protein droplets could nucleate
actin filaments. However, it remains unknown how LAT-ZAP70 phase separation affects signal
transduction in living cells.
 Here, I propose to determine whether synthetic phase separation of LAT-ZAP70 is sufficient to
activate the signaling pathways associated with antigen binding in both non-Tcells as well as Jurkat
cells. To do so, this project will utilize recently developed optogenetic tools that cause proteins to phase
separate when stimulated with light. Preliminary results from my studies suggest that synthetically
inducing LAT-ZAP70 protein phase separation with these optogenetic tools does indeed activate
downstream signaling pathways in non-T cells, but that causing LAT and ZAP70 to interact in other ways,
such as protein heterodimerization, does not activate these same pathways. To understand how protein
phase separation activates these signaling pathways, I will develop a two-color optogenetic system that
causes LAT-ZAP70 to heterodimerize and/or phase separate in a single cell. Thus, I can compare
how the same number of molecules interacting as heterodimers or liquid droplets activates signaling
pathways. I will also compare how well heterodimers or droplets activate signaling pathways in the
presence of T-cell specific factors, including the membrane phosphatase CD45, a proposed negative
regulator of LAT-ZAP70 complex activity. Finally, I plan to determine whether light-induced phase-
separation of LAT-ZAP70 in Jurkat T cells results in the secretion of cytokines and other key outcomes of T
cell activation. Overall, this project will determine whether and how protein phase separation of LAT-
ZAP70 is sufficient to activate the full T cell signaling response.

## Key facts

- **NIH application ID:** 9990525
- **Project number:** 5F31AI145218-02
- **Recipient organization:** PRINCETON UNIVERSITY
- **Principal Investigator:** Elliot Dine
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $28,650
- **Award type:** 5
- **Project period:** 2019-08-01 → 2020-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9990525

## Citation

> US National Institutes of Health, RePORTER application 9990525, Using optogenetics to dissect the role of signaling complex formation and organization in T-Cell activation (5F31AI145218-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9990525. Licensed CC0.

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