A new probe for super-resolution imaging of membranes elucidates trafficking pathways

Abstract

mCLING is a novel membrane probe for the study of membrane trafficking with demonstrated value in both live and fixed cells across a wide range of biological systems.

Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins

“…Dysferlin and myoferlin have also been identified in GM130‐positive Golgi compartments in primary lung epithelia and cancer cell lines (myoferlin) , and in cytoplasmic puncta in mouse myoblasts and human breast cancer cell lines ; consistent with the endosomal localization we observe, although endosomal markers were not specifically studied. Endogenous otoferlin co‐localizes with the trans‐Golgi t‐SNARE syntaxin‐16 in inner hair cells . Otoferlin expressed in HEK293 cells co‐localizes with the trans‐Golgi Rab8b , consistent with our study.…”

Mechanoresponsive lipid-protein nanoglobules facilitate reversible fibre formation in velvet worm slime

“…consists primarily of large, disordered proteins and several fatty acid variants 11, 12 . To reconcile this with our current observation of nanoglobule-based fibrillation, we performed sub-micron resolution compositional investigation using stimulated emission depletion (STED) microscopy by staining crude slime with a lipophilic probe (m-Cling) 21 and an isothiocyanate protein stain (Rhodamine b). STED imaging revealed the co-localization of lipid and protein signals in the nanoglobules (Fig.…”

Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation

“…Similar concerns with respect to the nature of the cycling membrane hold true for the use of FM dyes as reporters for presynaptic membrane turnover. However, when combined with photoconversion and EM (or super-resolution microscopy in case of the newly developed membrane-binding fluorophore mCLING) (Revelo et al, 2014) these probes allow the visualization and tracing of the internalized membrane on a timescale of seconds. PHluorins enable the monitoring of the activity-induced recycling of a specific SV protein, but only do so indirectly as their fluorescence reports on the pH environment, not on exo-or endocytosis per se.…”

“…Progress will depend on the further development of techniques to control synaptic activity and to monitor SV recycling in real-time. These techniques include combining optogenetic stimulation with genetic manipulations and optical imaging or rapid high pressure freezing EM as well as novel approaches to tag SV membranes during their exo-endocytotic itinerary independent of associated pH changes (Revelo et al, 2014). While the long half-life of SV proteins (days) (Cohen et al, 2013) is compatible with their recycling through multiple rounds of exo-endocytosis, it is likely that quality control mechanisms exist to ensure that SV proteins incorporated into reformed SVs are fully functional, whereas dysfunctional proteins are targeted for degradation.…”