Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30–300 nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates Ω-profile merging by providing sufficient plasma membrane tension to shrink the Ω-profile in neuroendocrine chromaffin cells containing ∼300 nm vesicles. Actin-directed compounds also induce Ω-profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate Ω-profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying Ω-profile merging.

Acidic pH inside cells indicates cellular dysfunctions such as cancer. Therefore, the development of optical pH sensors for measuring and imaging intracellular pH is a demanding challenge. The available pH-sensitive probes are vulnerable to e.g. photobleaching or autofluorescence background in biological materials. Our approach circumvents these problems due to near infrared excitation and upconversion photoluminescence. We introduce a nanosensor based on upconversion resonance energy transfer (UC-RET) between an upconverting nanoparticle (UCNP) and a fluorogenic pH-dependent dye pHrodo™ Red that was covalently bound to the aminosilane surface of the nanoparticles. The sensitized fluorescence of the pHrodo™ Red dye increases strongly with decreasing pH. By referencing the pH-dependent emission of pHrodo™ Red with the pH-insensitive upconversion photoluminescence of the UCNP, we developed a pH-sensor which exhibits a dynamic range from pH 7.2 to 2.5. The applicability of the introduced pH nanosensor for pH imaging was demonstrated by imaging the two emission wavelengths of the nanoprobe in living HeLa cells with a confocal fluorescence microscope upon 980 nm excitation. This demonstrates that the presented pH-nanoprobe can be used as an intracellular pH-sensor due to the unique features of UCNPs: excitation with deeply penetrating near-infrared light, high photostability, lack of autofluorescence and biocompatibility due to an aminosilane coating.

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.