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A Bcl-xL–Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

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Abstract

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, which is dependent on mitochondrial ATP. The anti-apoptotic Bcl-2 family protein Bcl-xL also regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, that Bcl-xL directly regulates endocytic vesicle retrieval in hippocampal neurons through protein–protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL–Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity.

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Figure 1: Bcl-xL overexpression enhances the rate of release of styryl dyes in hippocampal neurons.
Figure 2: Endogenous Bcl-xL participates in normal vesicle pool dynamics.
Figure 3: Bcl-xL increases the rate of mitochondrial ATP-resistant early endocytosis.
Figure 4: Calmodulin-dependent Bcl-xL translocation to synaptic vesicle membranes in stimulated neurons.
Figure 5: Drp1 is co-localized with clathrin and Mff on synaptic vesicles.
Figure 6: Bcl-xL and Drp1 co-localize with synaptophysin on synaptic vesicles.
Figure 7: Drp1 is required for formation of normal endocytic vesicles.
Figure 8: Mutations in the BH2 domain of Bcl-xL disrupt physical and functional interaction with Drp1.

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Acknowledgements

The authors wish to acknowledge L. Kaczmarek for thoughtful discussions of the data. The authors also thank G. Meisenbock (Center for Neural Circuits and Behaviour University of Oxford, UK) for providing synaptopHluorin for the sPH studies.

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Contributions

H.L. performed experiments, analyses and intellectual contributions; K.N.A. prepared novel reagents, performed experiments, analyses, intellectual contributions and prepared figures; E.L. assisted K.N.A. in preparing reagents and performing experiments; N.M. performed experiments and analyses; A.J. performed experiments and analyses; P.Z. performed experiments and analyses; P.L. prepared novel reagents for experiments; M.G. performed EM experiments; T.U. prepared novel reagents and performed experiments and analyses; J.G. performed experiments; C.R. supervised EM experiments; R.S.D. supervised work performed by P.L.; R.S.M. provided intellectual contribution and supervised experiments performed by T.U.; E.A.J. performed analyses, wrote the manuscript and provided intellectual contributions for, supervision of, and conception and planning of the project.

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Correspondence to Elizabeth A. Jonas.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Further fluorescence analysis for FM 5-95 experiments.

A. Maximum fluorescence values minus the minimum fluorescence values for all experiments in Figs 1 and 2 (***P<0.0001; **P = 0.0077; *P = 0.04). B. Normalized fluorescence change in 90 mM KCl ×3 (5 min rest in normal Tyrode’s wash medium in between each stimulus); N = 9 GFP ctl puncta, 9 GFP-Bcl-xL puncta, 3 independent coverslips were used for each group. Bleaching curve shows normalized fluorescence change in the absence of stimulation. Statistics are represented as mean+/-S.E.M.

Supplementary Figure 2 Transfection of shRNA constructs decreases Bcl-xL, Drp1 and calmodulin protein levels. Purity of subcellular fractions of hippocampal lysate.

A. Immunoblots of lysate of SHSY5Y cells expressing Bcl-xL shRNA or Bcl-xL shRNA in combination with Bcl-xL shRNA resistant constructs. B. (left panel) Immunoblots of lysate of hippocampal neurons expressing scrambled shRNA or Bcl-xL shRNA. (right panel) Immunoblots of lysate of hippocampal neurons overexpressing or not GFP-Bcl-xL. C. Immunoblots with indicated antibodies performed on lysate of hippocampal neurons expressing calmodulin shRNA or scrambled shRNA. D. (Left panel) Immunoblots of subcellular fractions using the indicated antibodies of non-stimulated cultured hippocampal neuron lysate. (Right panel) Immunoblots of subcellular fractions using the indicated antibodies of cultured hippocampal neuron lysate from neurons stimulated with 90 mM KCl for 90 s. E. Immunoblots of subcellular fractions (as indicated) of cultured hippocampal neuron lysate using the indicated antibodies. Left lane shows cell lysate. F. Fluorescent, phase and overlay images of cultured hippocampal neurons transduced with lentivirus construct for GFP-Drp1 shRNA. 100% of cells are transduced. Scale bar, 100 μm. G. Immunoblots of Drp1 shRNA knockdown and overexpression of Drp1 shRNA resistant constructs in 293T cells. H. Immunoblots of lysate of cortical neurons expressing Drp1 shRNA at indicated concentrations. I. Immunogold labeling of Drp1 in scrambled control and Drp1 shRNA-expressing cultured hippocampal neurons (N = 13micrographs for scrambled control, N = 21micrographs for Drp1 shRNA; ***p<0.0001). The average number of synapses in each micrograph was not different between the two groups (1.42 ± 0.14 for Drp shRNA, 1.3 ± 0.13 for scrambled control). Statistics are represented as mean+/-S.E.M.

Supplementary Figure 3 Drp1 localizes to mitochondria and anti-Drp1 and anti-Dynamin I/II antibodies are specific.

A. Immuno-electron micrograph shows localization of Drp1-labeled particles to mitochondria. B. Immunoblots for Drp1 of indicated sub-fractions of cell lysates of cultured unstimulated hippocampal neurons or cultured hippocampal neurons stimulated with 90 mM KCl for 90 s with or without CaMi. GAPDH serves as protein control for cytosolic protein amount, COX IV as control for mitochondrial protein amount, synaptotagmin as control for synaptic vesicle membrane protein amount. C. Immunoblot of rat brain lysate probed with the indicated antibodies.

Supplementary Figure 4 Non-normalized fluorescent data for synaptopHluorin experiments before and after bafilomycin.

A. SynaptopHluorin experiments comparing effects of bafilomycin on GFP-Bcl-xL over-expressing and GFP expressing neurons. B. SynaptopHluorin experiments comparing effects of bafilomycin on Bcl-xL shRNA-expressing cells and on neurons expressing scrambled control shRNA.

Supplementary Figure 5 Uncropped images of films for the key experiments in the main figures.

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Li, H., Alavian, K., Lazrove, E. et al. A Bcl-xL–Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nat Cell Biol 15, 773–785 (2013). https://doi.org/10.1038/ncb2791

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