Building synthetic cells performing calcium-triggered membrane fusion

Introduction:: In cells, membrane fusion is a key mechanism that underpins exocytosis and is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated in synthetic systems, few can respond to external stimuli. A stimuli-responsive membrane fusion scheme could enable controlled synthetic exocytosis in synthetic cells, which will allow for the engineering of smart biosensors that sense cues and then precisely deliver versatile payloads. In response to this gap in technology, we developed a calcium-triggered membrane fusion strategy building upon existing DNA-mediated membrane fusion technology.


Materials and Methods:: We assembled phospholipid vesicles of two distinct sizes: small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs). In all experiments, SUVs and GUVs are membrane conjugated with complementary single-strand DNA oligonucleotides. Membrane interactions between SUVs and GUVs were tested by mixing solutions containing each vesicle type together in bulk. To inhibit fusion, surface-bound PEG chains 1, 2, & 5 kDa in length were tested. Later, a custom peptide construct (Chol-CCS-PEG_2K) was synthesized where a calpain cleavage site (CCS) is conjugated to a cholesterol molecule on one side and a 2 kDa PEG chain on the other side. For experiments with Chol-CCS-PEG_2K, purified calpain-1 and CaCl₂ were used. Furthermore, a novel membrane fusion assay was designed to convincingly prove membrane fusion. In this assay, GUVs were encapsulated with His-tagged blue fluorescent protein and SUVs were made with lipids containing nickel-NTA, which has an affinity to His-tag. Consequently, membrane fusion between SUVs and GUVs will result in SUV membranes incorporated into the GUV membrane. Subsequently, His-tagged proteins will translocate to the membrane. Lastly, for testing SUV-GUV fusion in a vesicle-in-vesicle system (i.e., SUVs inside GUV), SUVs conjugated with DNA and Chol-CCS-PEG_2K were encapsulated inside GUVs. In addition, complementary DNA strands and an excess amount of Chol-CCS-PEG_2K were also encapsulated inside the GUVs, which then become conjugated to the inner membrane of GUVs.


Results, Conclusions, and Discussions:: DNA-mediated membrane interactions between SUVs and GUVs were first successfully established. Next, surface-bound PEG chains of various sizes were added to both SUV and GUV membranes. We found conjugating PEG­­_2K (2 kDa) on opposing membranes was the minimal polymer size to effectively inhibit membrane interactions. However, PEG_2K on either membrane was not sufficient to block membrane interactions. Chol-CCS-PEG_2K was then conjugated to membrane surfaces and membrane interactions were only observed in the presence of both calcium and calpain-1. Perceived membrane interactions were then demonstrated to be the occurrence of membrane fusion. Specifically, using our membrane fusion assay, binding of His-tagged fluorescent protein to the membrane was only observed in the presence of both calcium and calpain-1. With calcium-triggered membrane fusion strategy validated, we then encapsulated SUVs and calpain-1 in GUVs to develop synthetic exocytosis. Without PEG chains on opposing membranes, membrane interactions were observed. With Chol-CCS-PEG_2K conjugated to the outer membrane of SUVs and inner membrane of GUVs, no membrane interactions were observed. However, membrane interactions were observed when calcium ions were shuttled into the GUV via ionophores, which suggests cleavage of PEG and subsequent occurrence of exocytosis-like behavior.

In summary, we have developed a strategy that mimics in vivo SNARE-mediated membrane fusion by recapitulating its calcium-dependent nature. In addition, we integrated this strategy in a vesicle-in-vesicle system and demonstrated evidence of controlled synthetic exocytosis upon shuttling calcium ions into the GUV. Moving forward, the synthetic cell can be functionalized with mechanosensing mechanisms, for example by reconstitution of mechanosensitive channels on GUV membranes. Such systems would have a significant impact on synthetic cell research by introducing controlled synthetic exocytosis only when calcium, a common secondary messenger molecule, enters the synthetic cell, triggered by a mechanical stimulus. With this development, the generation of synthetic cell-based biosensors that secrete a designated payload in response to mechanical stimuli can be realized. Furthermore, by utilizing synthetic exocytosis, communications between synthetic cells and natural cells can be established, which will enable a new way to study cellular behaviors and disease progression with more precision.


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