SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals.

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

Biomolecular actuators for genetically selective acoustic manipulation of cells.

The ability to physically manipulate specific cells is critical for the fields of biomedicine, synthetic biology, and living materials. Ultrasound has the ability to manipulate cells with high spatiotemporal precision via acoustic radiation force (ARF). However, because most cells have similar acoustic properties, this capability is disconnected from cellular genetic programs. Here, we show that gas vesicles (GVs)-a unique class of gas-filled protein nanostructures-can serve as genetically encodable actuators for selective acoustic manipulation. Because of their lower density and higher compressibility relative to water, GVs experience strong ARF with opposite polarity to most other materials. When expressed inside cells, GVs invert the cells' acoustic contrast and amplify the magnitude of their ARF, allowing the cells to be selectively manipulated with sound waves based on their genotype. GVs provide a direct link between gene expression and acoustomechanical actuation, opening a paradigm for selective cellular control in a broad range of contexts.

Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria.

Ultrasound allows imaging at a much greater depth than optical methods, but existing genetically encoded acoustic reporters for in vivo cellular imaging have been limited by poor sensitivity, specificity and in vivo expression. Here we describe two acoustic reporter genes (ARGs)-one for use in bacteria and one for use in mammalian cells-identified through a phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea that provide stronger ultrasound contrast, produce non-linear signals distinguishable from background tissue and have stable long-term expression. Compared to their first-generation counterparts, these improved bacterial and mammalian ARGs produce 9-fold and 38-fold stronger non-linear contrast, respectively. Using these new ARGs, we non-invasively imaged in situ tumor colonization and gene expression in tumor-homing therapeutic bacteria, tracked the progression of tumor gene expression and growth in a mouse model of breast cancer, and performed gene-expression-guided needle biopsies of a genetically mosaic tumor, demonstrating non-invasive access to dynamic biological processes at centimeter depth.

Sec17 (α-SNAP) and an SM-tethering complex regulate the outcome of SNARE zippering in vitro and in vivo.

Zippering of SNARE complexes spanning docked membranes is essential for most intracellular fusion events. Here, we explore how SNARE regulators operate on discrete zippering states. The formation of a metastable trans-complex, catalyzed by HOPS and its SM subunit Vps33, is followed by subsequent zippering transitions that increase the probability of fusion. Operating independently of Sec18 (NSF) catalysis, Sec17 (α-SNAP) either inhibits or stimulates SNARE-mediated fusion. If HOPS or Vps33 are absent, Sec17 inhibits fusion at an early stage. Thus, Vps33/HOPS promotes productive SNARE assembly in the presence of otherwise inhibitory Sec17. Once SNAREs are partially zipped, Sec17 promotes fusion in either the presence or absence of HOPS, but with faster kinetics when HOPS is absent, suggesting that ejection of the SM is a rate-limiting step.

Sec17/Sec18 act twice, enhancing membrane fusion and then disassembling cis-SNARE complexes.

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.