We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid “tetrazine-click” reaction into Cer-SiR, a highly photostable “vital dye” that enables prolonged live-cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is nontoxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.

The cell interior is a complex and demanding environment. An incredible variety of molecules jockey to identify the correct position – the specific interactions that promote biology, which are often hidden among countless unproductive options. Ensuring that the business of the cell is successful requires sophisticated mechanisms to impose temporal and spatial specificity – both on transient interactions and their eventual outcomes. Two strategies employed to regulate macromolecular interactions in a cellular context are colocalization and compartmentalization. Macromolecular interactions can be promoted and specified by localizing the partners within the same subcellular compartment, or by holding them in proximity through covalent or non-covalent interactions with proteins, lipids, or DNA – themes that are familiar to any biologist. The net result of these strategies is an increase in effective molarity: the local concentration of a reactive molecule near its reaction partners. We will focus on this general mechanism, employed by nature and adapted in the lab, which allows delicate control in complex environments: the power of proximity to accelerate, guide, or otherwise influence the reactivity of signaling proteins and the information that they encode.

We reported recently that certain β³-peptides self-assemble in aqueous solution into discrete bundles of unique structure and defined stoichiometry. The first β-peptide bundle reported was the octameric Zwit-1F, whose fold is characterized by a well-packed, leucine-rich core and a salt-bridge-rich surface. Close inspection of the Zwit-1F structure revealed four nonideal interhelical salt-bridge interactions whose heavy atom–heavy atom distances were longer than found in natural proteins of known structure. Here we demonstrate that the thermodynamic stability of a β-peptide bundle can be enhanced by optimizing the length of these four interhelical salt bridges. Combined with previous work on the role of internal packing residues, these results provide another critical step in the “bottom-up” formation of β-peptide assemblies with defined sizes, reproducible structures, and sophisticated function.