Overview

In mammalian synthetic biology, most gene regulatory systems operate at the transcriptional level, where external signals modulate promoter activity to control gene expression. While effective, transcriptional switches often exhibit slow response dynamics, as they depend on transcription, translation, folding, and secretion of newly synthesized proteins—a process that can take several hours.1

In contrast, post-translational control systems acting directly on protein secretion can respond within minutes. However, existing secretion-based systems are often restricted to specific cell types (such as neuroendocrine cells) and rely on ER-based protein retention, which can cause ER stress and reduce system efficiency.2

To address these limitations, we designed SPARK (Signal-Programmable Activation of Regulated seCretion), a modular and universal secretion control platform that regulates the release of preformed secretory vesicles through signal-induced protein dissociation.

By bypassing transcriptional delays and avoiding ER aggregation, SPARK enables fast, reversible, and tunable secretion across diverse mammalian chassis cells.

System Architecture

SPARK is organized into three modular layers, Release, Response, and Retention, that can be independently optimized or exchanged to interface with a wide variety of input signals (light, drugs, metabolites, etc.) and output proteins (enzymes, hormones, reporters).(Figure 1C).

Design of the SPARK system

Figure 1. Conceptual design of the SPARK system.

(A) Modular architecture composed of Release, Response, and Retention modules. (B) Secretory vesicles are anchored via LifeAct and released upon signal induction. (C) Modular design allows diverse input–output combinations.

Release Module

The Release Module defines how the target protein (POI) enters the secretory pathway and is prepared for signal-controlled release.

Components:

  • Signal Peptide (IgK): Directs nascent proteins into the endoplasmic reticulum, initiating vesicle formation and ensuring proper secretion routing.

  • Gene of Interest (GOI): The user-defined output protein; in our characterization, NanoLuc luciferase served as a quantitative reporter.

  • Furin Cleavage Site (FS): Recognized and cleaved by endogenous furin enzymes in the Golgi, releasing the GOI into the vesicle lumen while leaving the transmembrane anchor in place.

  • Transmembrane Domain (TMD): Anchors the fusion protein to the vesicle membrane and positions downstream elements (e.g., NS3a or light-responsive domains) on the cytosolic side for interaction with the Response and Retention modules.

Through this arrangement, proteins are packaged into vesicles and held in a secretion-ready state, awaiting signal activation.

Response Module: Signal-Specific Dissociation

The Response Module determines which input signal triggers vesicle release. It consists of a pair of separable proteins that link the vesicle membrane (via TMD) to the cytoskeleton (via LifeAct) before stimulation. Upon receiving the defined signal, the protein pair dissociates, freeing the vesicle for exocytosis.

Because this module is interchangeable, SPARK can be adapted to respond to diverse stimuli simply by swapping the signal-responsive protein pair.

Examples implemented in our project (see our Engineering Page and Results Page for more detail):


• Light-inducible systems:

mMaple3 (violet-light photolytic protein)3

CarH (green-light dissociation via vitamin B12 photolysis)4

BphP1–PpsR2 (near-infrared photodissociation pair)5

• Chemically inducible system:

NS3a–ANR pair, which dissociates rapidly upon Grazoprevir exposure6,7


Each pair can be modularly replaced by other well-characterized inducible interactions (e.g., rapamycin-, calcium-, or temperature-responsive pairs), making SPARK a plug-and-play framework for various sensing contexts.

Response module mechanism

Figure 2.Response Module logic: signal-induced dissociation releases pre-anchored vesicles.

Retention Module: Vesicle Anchoring via the Cytoskeleton

The Retention Module secures secretory vesicles at defined subcellular positions, preventing constitutive secretion. SPARK employs LifeAct, a 17-amino-acid peptide that binds F-actin with high specificity while minimally perturbing actin dynamics.8

In the resting state, LifeAct tethers vesicles via the Response Module to the cytoskeleton, effectively storing secretion cargo at the ready. Upon signal input, the dissociable protein pair detaches from LifeAct, releasing vesicles for rapid exocytosis.

This strategy enables precise spatial control of vesicle storage without imposing ER stress or requiring specialized secretory machinery.

Advantages of the Modular SPARK Framework

Feature Description Example Implementation
Universal chassis compatibility Operates in standard mammalian cell lines (e.g., HEK293T, HeLa) without specialized secretory organelles. chemSPARK, optoSPARK
Rapid response Acts at the secretion level, bypassing transcription/translation delays. 40×secretion within 3 h
Flexible input design Response Module can be replaced to respond to light, drugs, or metabolic cues. NS3a-ANR / BphP1-PpsR2 / CarH
Tunable output Output protein determined by the GOI in the Release Module. NanoLuc, insulin, growth factors

Expanding the SPARK Platform

Because all modules follow a standardized interface design (TMD-Response-LifeAct architecture), SPARK can be easily reprogrammed to sense new inputs or control alternative outputs:

Input Expansion:

Replace the Response Module with other dissociation-based systems (e.g., Ca2+-binding domains, small-molecule disruptors, protease cleavage pairs).

Output Expansion:

Substitute NanoLuc with therapeutic or signaling proteins (e.g., insulin, cytokines, neuropeptides).

Feedback Integration:

Couple SPARK to intracellular sensors to create closed-loop control circuits, enabling autonomous homeostasis (e.g., calcium-responsive insulin release).

Thus, SPARK serves not only as a proof-of-concept for fast secretion control but also as a generalizable chassis for building next-generation post-translational gene circuits.

References

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[1] Xie, M. & Fussenegger, M. Designing cell function: assembly of synthetic gene circuits for cell biology applications. Nat Rev Mol Cell Biol 19, 507-525 (2018). https://doi.org/10.1038/s41580-018-0024-z
[2] Wang, X. et al. A programmable protease-based protein secretion platform for therapeutic applications. Nat Chem Biol 20, 432-442 (2024). https://doi.org/10.1038/s41589-023-01433-z
[3] Hoi, H. et al. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J Mol Biol 401, 776-791 (2010). https://doi.org/10.1016/j.jmb.2010.06.056
[4] Padmanabhan, S., Jost, M., Drennan, C. L. & Elias-Arnanz, M. A New Facet of Vitamin B(12): Gene Regulation by Cobalamin-Based Photoreceptors. Annu Rev Biochem 86, 485-514 (2017). https://doi.org/10.1146/annurev-biochem-061516-044500
[5] Bellini, D. & Papiz, M. Z. Structure of a bacteriophytochrome and light-stimulated protomer swapping with a gene repressor. Structure 20, 1436-1446 (2012). https://doi.org/10.1016/j.str.2012.06.002
[6] Failla, C., Tomei, L. & De Francesco, R. An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A. J Virol 69, 1769-1777 (1995). https://doi.org/10.1128/JVI.69.3.1769-1777.1995
[7] Cunningham-Bryant, D. et al. A Chemically Disrupted Proximity System for Controlling Dynamic Cellular Processes. J Am Chem Soc 141, 3352-3355 (2019). https://doi.org/10.1021/jacs.8b12382
[8] Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods 5, 605-607 (2008). https://doi.org/10.1038/nmeth.1220