The release ofhormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high- resolution patch -clamp capacitance technique, we studied single vesicles and asked whether the Secl/Muncl8 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Muncl8-1 mutants were transfected into lactotrophs to affect the interaction of Muncl8-1 with syntaxinl (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Muncl8-1 E466K increased the frequency ofthe fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion oflarger vesicIes, since overexpression of Munc18-1 R39C did not affect the ave rage size of vesicIes, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binarytrans-complexes. However, neither form ofMuncl8-1 affected the interaction ofSyb2 with the preformed binary cis-Synt1ASNAP25B complexes. Thisindicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing ves ici es solely to Synt1 at the plasmalemma andfavoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Muncl8-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Muncl8-1 interactions with Synt1A, Rab3A, and Mints.
COBISS.SI-ID: 28521433
Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolicfree glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600mu. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with theone recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration inadipocytes by a factor of 3.6; however, we recorded no effect on delta ratio(DeltaR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p ( 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.
COBISS.SI-ID: 28263129