Much insight into biological function has come from the development of new biophysical techniques and instrumentation development, driven by important specific biological questions.
After a formal training in physics I chose to study the function of the visual pigment rhodopsin. After light absorption intramolecular charge movements give rise to a major conformational change that allows activation of an enzymatic cascade inside the cell, eventually leading to a change in the rate of neurotransmitter release. I developed a new method to spread the native rhodopsin containing cellular membranes on a filter (Lindau et al., 1980) such that direct electrical measurements could be made and the charge movement mechanism could be characterized.
Fascinated by exploring cellular and molecular mechanisms in biology using biophysical methods, I had the privilege to work as a post-doc with Nobel Laureate Erwin Neher in 1984/85. In his lab I learned patch clamping and worked on secretory cells. To determine if ion channels play a role in stimulation of histamine release from mast cells I developed a novel patch-clamp configuration which we named "slow whole cell" where electrical access to the cell was not by patch disruption but by creating small pores in the patch under the pipette such that the biochemistry inside was minimally disturbed (Lindau and Fernandez, 1986). This was the basis of what is now called "permeabilized patch" recordings.
Since then, my activities were mainly driven by an interest in the mechanisms of exocytosis and transmitter release, which represent one of the most exciting topics in cell biology, neurobiology and biophysics. The process of regulated exocytosis is responsible for release of neurotransmitters and neuropeptides by nerve terminals and endocrine cells, release of enzymes or cytotoxic proteins by granulocytes, or release of histamine and other mediators by mast cells. During exocytosis the membrane of secretory granules fuses with the plasma membrane of the cell thereby releasing their contents through the fusion pore. Although biochemical studies revealed a set of proteins that are essential for exocytosis, the specific molecular mechanisms of fusion are still obscure. As for ion channels, functional studies of the fusion processes are essential to elucidate the molecular mechanisms of fusion.
We investigate single exocytotic fusion events by measurements of membrane capacitance using the patch clamp technique exploiting the fact that exocytosis and endocytosis are associated with changes in plasma membrane area leading to proportional changes of electrical membrane capacitance (Lindau, 1991; Lindau and Neher, 1988). We developed an improved method that allows to investigate single vesicle exocytosis and fusion pore dynamics by patch capacitance measurements (Debus and Lindau, 2000; Lindau, 2012). The method allows for the investigation of the opening of single exocytotic fusion pores in neurosecretory vesicles with a resolution similar to that obtained in conventional single channel recordings. In addition, release of oxidizable substances from single vesicles can be monitored by amperometric techniques. To investigate directly the relation between fusion pore dynamics and transmitter release in neuronal cell types we developed the method of patch amperometry, which combines high resolution patch capacitance measurements with amperometric detection of transmitter release inside the patch pipette (Dernick et al., 2007; Dernick et al., 2005).
To understand the mechanism of vesicle fusion we developed microfabricated electrode arrays that allow measurement of release events with simultaneous fluorescence imaging using total internal reflection fluorescence excitation of fluorescent probes on the cell surface (Dias et al., 2002; Hafez et al., 2005; Kisler et al., 2012).
Besides of the technical developments in this area, we applied these methods to advance our understanding of the mechanisms of biological membrane fusion and its regulation. We published the first characterization of exocytotic fusion pore opening in neurosecretion (Albillos et al., 1997; Dernick et al., 2003; Gong et al., 2007). We discovered important roles for the C termini of the SNARE proteins SNAP-25 and synaptobrevin in the fusion mechanism (Fang et al., 2008; Ngatchou et al., 2010). Other important steps are the tethering and docking events which vesicles undergo preceding the actual fusion event. We succeeded in measuring for the first time the forces tethering vesicles together using horse eosinophil granules (Valero et al., 2008).
Current research is focused on understanding the relation between conformational changes in the SNARE complex and fusion pore formation using experimental and computational approaches. We also develop an approach to characterize docking forces in vesicle-plasma membrane interactions using atomic force microscopy and develop a CMOS microchip device for amperometric high throughput characterization of fusion events.
References can be found in Publications