Amperometric Measurements at Cells Support a Role for Dynamin in the Dilation of the Fusion Pore during Exocytosis

Abstract

Dynamin is a GTPase mechanochemical enzyme involved in the late steps of endocytosis, where it separates the endocytotic vesicle from the cell membrane. However, recent reports have emphasized its role in exocytosis. In this case, dynamin may contribute to the control of the exocytotic pore, thus suggesting a direct control on the efflux of neurotransmitters. Dynasore, a selective inhibitor of the GTPase activity of dynamin, was used to investigate the role of dynamin in exocytosis. Exocytosis was analyzed by amperometry, thus revealing that dynasore inhibits exocytosis in a dose-dependent manner. Analysis of the exocytotic peaks showed that the inhibition of the GTPase activity of dynamin led to shorter, smaller events. This observation, together with the rapid effect of dynasore, suggests that the blocking of the GTPase induces the formation of a more narrow and short-lived fusion pore. These results suggest that the GTPase properties of dynamin are involved in the duration and kinetics of exocytotic release. Interestingly, and in strong contrast with its role in endocytosis, the mechanochemical properties of dynamin appear to contribute to the dilation and stability of the pore during exocytosis.

How “Full” is “Full Fusion” during Exocytosis from Dense Core Vesicles? Effect of SDS on “Quantal” Release and Final Fusion Pore Size

“…As discussed elsewhere 35 this leads to a variety of situations depending on the value of the ratios k slow /k diff ρ and k slow /k fast . Though, only one of these possibilities is of interest here since it leads to the observation of a slow second exponential mode (τ slow = 1/k slow ) following the fast first one (τ = 1/k diff ρ ), as also observed by Ewing et al: 31,32 …”

“…The majority (84% of the spikes) displayed descending branches that required two-exponential fits to be modeled, 31,32,35 viz., i decay fit (t) ∝ exp(−t/τ) + 0.12 × exp(−t/τ slow ), with τ / ms = 1.1 (0.9; 1.6), τ slow / ms = 5.4 (3.9; 7.3) (see Figure 7).…”

“…Provided the value of the fusion pore radius, R pore , is small, viz., such as R pore /R ves < 0.4, [26][27][28] a condition that largely encompasses all the situations considered in this work, ρ is very closely approximated by ρ = R pore /(π 2 R ves ), [26][27][28] so that k 31,32 Indeed, the observation of amperometric spikes with exponential decay branches is tantamount to saying that R pore has achieved a constant value after the spikes maxima, 26,27 so that k diff ρ is constant. This is achieved at different specific times depending on the rate of the fusion pore expansion.…”

“…33 Several effects may be invoked to rationalize such observation. A first one, put forward by Ewing and colleagues [31][32][33] consists in considering that the fusion pore closes before complete release could occur. However, even if several cases in which this certainly occurs have been reported by the same group, this cannot be general.…”

‘Full fusion’ is not ineluctable during vesicular exocytosis of neurotransmitters by endocrine cells

“…Although manageable with difficulty by non-experts due to the requirement for human decision at several critical stages [23,54,55], such simulations established quantitatively for the first time that most amperometrically detected events involve fusion pores whose expansion was stalled at a maximum size of only approximately one-tenth of that of the vesicle. These results, as well as other evidence inferred by others based on purely experimental strategies [30,31,[66][67][68][69], have instilled the concept that, in endocrine cells, most releasing events proceed entirely through fusion pores whose radii remain much smaller than those of vesicles, i.e. they contradict the 'full fusion' paradigm.…”