Of the techniques currently available to monitor dense core granule exocytosis in adrenal chromaffin cells, two have proven particularly useful carbon-fiber amperometry and total internal reflection fluorescence (TIRF) microscopy. Amperometry enables the detection of oxidizable catecholamines escaping a fusion pore with millisecond time resolution. TIRF microscopy, and its variant polarized-TIRF (pTIRF) microscopy, provides information on the characteristics of fusion pores at temporally later stages. Used in conjunction, amperometry and TIRF microscopy allow an investigator to follow the fate of a fusion pore from its formation to expansion or reclosure. The properties of fusion pores, including their structure and dynamics, have been shown by multiple groups to be modified by the dynamin GTPase (Dyn1). In this chapter, we describe how amperometry and TIRF microscopy enable insights into dynamin-dependent effects on exocytosis in primary cultures of bovine adrenal chromaffin cells.Membrane fusion and fission are indispensable parts of intracellular membrane recycling and transport. Electrophysiological techniques have been instrumental in discovering and studying fusion and fission pores, the key intermediates shared by both processes. In cells, electrical admittance measurements are used to assess in real time the dynamics of the pore conductance, reflecting the nanoscale transformations of the pore, simultaneously with membrane leakage. Here, we described how this technique is adapted to in vitro mechanistic analyses of membrane fission by dynamin 1 (Dyn1), the protein orchestrating membrane fission in endocytosis. We reconstitute the fission reaction using purified Dyn1 and biomimetic lipid membrane nanotubes of defined geometry.  https://www.selleckchem.com/products/envonalkib.html  We provide a comprehensive protocol describing simultaneous measurements of the ionic conductance through the nanotube lumen and across the nanotube wall, enabling spatiotemporal correlation between the nanotube constriction by Dyn1, leading to fission and membrane leakage. We present examples of "leaky" and "tight" fission reactions, specify the resolution limits of our method, and discuss how our results support the hemi-fission conjecture.Dynamin-related proteins on both the mitochondrial outer and inner membranes mediate membrane fusion. Mitochondrial fusion is regulated in many different physiological contexts including cell cycle progression, differentiation pathways, stress responses, and cell death. Mitochondrial fusion is opposed by mitochondrial division and requires movement of mitochondria on microtubules. We developed a cell-free reconstituted mitochondrial fusion assay to circumvent the complexity of the pathways impinging on the activity of the mitochondrial fusion machinery in vivo. This allows for quantification of mitochondrial fusion in defined conditions and in the absence of other processes such as mitochondrial division or transport. The impact of proteins or small molecules on mitochondria fusion can also be assessed. Here we describe the cell-free mitochondrial fusion assay using mitochondria isolated from mouse embryonic fibroblasts.Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid-silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.A common feature of dynamin-related proteins (DRPs) is their use of guanosine triphosphate (GTP) to control protein dynamics. In the case of the endoplasmic- reticulum- (ER)-resident membrane protein atlastin (ATL), GTP binding and hydrolysis result in membrane fusion of ER tubules and the generation of a branched ER network. In this chapter, we describe two independent methods for dissecting the mechanism underlying nucleotide-dependent quaternary structure and conformational changes of ATL, focusing on size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and Förster resonance energy transfer (FRET), respectively. The high temporal resolution of the FRET-based assays enables the ordering of the molecular events identified in structural and equilibrium-based SEC-MALS studies. In combination, these complementary methods report on the oligomeric states of a system at equilibrium and timing of key steps along the enzyme's catalytic cycle. These methods are broadly applicable to proteins that undergo ligand-induced dimerization and/or conformational changes.Microscale thermophoresis (MST ) is a robust new fluorescence-based technology that enables measurement of biomolecular interactions and binding affinities (KD). MST is an immobilization-free alternative to surface plasmon resonance (SPR ) and is cost-effective relative to isothermal titration calorimetry (ITC ). In this chapter, using Drp1 as an example, we demonstrate for the first time, the application of MST to the determination of DSP-lipid interactions and the accurate measurement of KD under physiologically relevant solution conditions.The human guanylate-binding protein 1 (hGBP1) is the best characterized isoform of the seven human GBPs belonging to the superfamily of dynamin-like proteins (DLPs). As known for other DLPs, hGBP1 also exhibits antiviral and antimicrobial activity within the cell. hGBP 1, like hGBPs 2 and 5, carries a CAAX motive at the C-terminus leading to isoprenylation in the living cells. The attachment of a farnesyl anchor and its unique GTPase cycle provides hGBP1 the ability of a nucleotide- stimulated polymerization and membrane binding. In this chapter, we want to show how to prepare farnesylated hGBP1 (hGBP1fn) by bacterial synthesis and by enzymatic modification, respectively, and how to purify the non-farnesylated, as well as the farnesylated hGBP1, by chromatographic procedures. Furthermore, we want to demonstrate how to investigate the special features of polymerization by a UV-absorption-based turbidity assay and the binding to artificial membranes by means of fluorescence energy transfer.