Reconstitution/nanomechanics of fusion

 

We used nanodiscs to isolate fusion pores in their initial open state. Using biochemical reconstitution, we demonstrated that the nascent fusion pore is a hybrid structure, formed by both lipids and the transmembrane domains (TMDs) of SNAREs. In this transient structure, one specific side of the SNARE TMDs face the lumen of the fusion pore (shown in red).

 

 

 

ND_BLM_figure

 

 ND (nanodisc)-BLM (Black Lipid Membrane) setup and typical recording of fusion pore dynamics. (a) The v-SNARE (synaptobrevin 2 or syb2), reconstituted in a ND, interacts with the t-SNAREs present in the BLM to form a fusion pore, as shown in the illustration. (b) Traces of single pores at ΔΨ = −50 mV for ND bearing ~ 5 copies of syb2. Closed (C) and open (O) states are indicated, along with the respective currents.

 

 

 

 

A primary focus of the lab concerns the mechanisms of action of Ca2+ sensors for exocytosis. Synaptotagmin-1, the Ca2+ sensor for synchronous neurotransmitter release, contains tandem C2 domains and triggers exocytosis via Ca2+-dependent insertion into lipid bilayers. This figure depicts Doc2β, a separate but related Ca2+ sensor for asynchronous and spontaneous neurotransmitter release. Using site-specific labeling with an environmentally sensitive probe, we have shown that Doc2, like syt1, penetrates membranes in a Ca2+-dependent manner. We have used a quantitative approach to measure membrane penetration depth and found that, in the case of Doc2β, the depth of membrane penetration depends specifically on a PIP2, a dynamically-regulated phospholipid present at release sites. These findings define biophysical diversity among these Ca2+ sensors and suggest new regulatory mechanisms for vesicular secretion.

 

 

 

 

DNA-nanostructure-templated protein reconstitution in nanodisc. A self-assembled DNA tetrahedron is first functionalized with proteins of interest (POI), and then reconstituted into a nanodisc. This method enables precise control of the copy number and orientation of the POI in the nanodisc, via customized design and steric hindrance of the DNA scaffold.

 

 

 

 

Nanodisc arrangement on DNA origami. A DNA origami structure (in this case a cylinder-like nanocage) is used to organize nanodiscs into 3D array, providing a unique platform to study protein-protein and protein-membrane interactions in a distance/stoichiometry-defined manner.

 

 

 

Giant vesicle-nanodisc dye leakage assay

We recently developed an assay to visualize pore formation in the membrane of giant unilamellar vesicles (GUVs). We encapsulate cargos of different sizes in the lumen of GUVs and generate pores in the membrane mediated by membrane binding proteins and/or SNARE bearing nanodiscs. With live, multi-channel Airyscan imaging, we can simultaneously monitor the movement of up to four different molecules through the pores ranging from ions to large protein complexes. In this example, the t-SNARE bearing GUV membrane is labelled with rhodamine-DOPE and contains encapsulated Alexa-647 dye. Upon addition of v-SNARE bearing nanodiscs, the nanodiscs bind the surface of the GUV, allowing trans-SNARE pairing and pore formation to release the dye.

 

 

Figure: AFM images of: (A) synaptotagmin (syt) 1 reconstituted into a lipid bilayer, (B) DNA tetrahedron tiles in liquid on mica, (C) phase separation of DOPC (darker) and DPPS (brighter) in a lipid bilayer in 1 mM free Ca2+ and (D) 16 nm nanodiscs in liquid on mica.

Summary: Our lab is using atomic force microscopy (AFM) to study the oligomerization state of reconstituted syt1 and the Ca2+ dependent phase separation of PC and PS lipid species. Furthermore, we use AFM as a means of quality control for the size and morphology of our fabricated nanodiscs and DNA nanostructures.