We sought to deduce the role of membrane lipid
We sought to deduce the role of membrane lipid determinants on endo- and exocytosis by applying a combination of metabolic (lipid) engineering approaches and biophysical methods, e.g. classical optical imaging technologies and high-resolution time-resolved patch-clamp/capacitance measurements. The latter method uses isolated protoplasts that were enzymatically freed from their cell wall in order to allow for freely accessing the plasma membrane. However, removing the cell wall eliminates the intracellular turgor pressure, which can reach greater than a megapascal (MPa) in fungal cells, and which might drastically influence membrane related processes . This raises the question whether protoplasts are physiologically active and display natural membrane trafficking. In a recent study, yeast protoplasts were shown to grow in an energy dependent manner and to exhibit endocytic and exocytic activity in capacitance recordings which was further confirmed by classical imaging technologies showing membrane trafficking [27,28].
We considered metabolically engineered yeast protoplasts as suitable system to study the role of lipid determinants on endo- and exocytosis by membrane capacitance recordings. This method measures changes to cell membrane area in real time with high temporal resolution thus providing detailed information on fundamental characteristics of endocytosis and exocytosis such as the frequency of vesicle fission and fusion as well as the size of endo- and exocytic vesicles.
Materials and methods
Conclusions Here we introduce a combination of metabolic engineering approaches for manipulating endogenous lipid biosynthesis pathways and electrophysiological membrane capacitance measurements in order to establish a system to study lipid determinants of eukaryotic cells for endo- and exocytosis in vivo. Systematic manipulation of both fatty 1215 unsaturation and sterol biosynthesis strongly affected endocytosis in yeast. In contrast, exocytosis was strongly perturbed upon low lipid unsaturation, whereas the cellular ergosterol content had almost no influence on the cell's exocytic activity. This work illustrates the requirement of defined lipid-mediated membrane characteristics for proper vesicle fusion and fission in the context of endo- and exocytosis. Thus, we show that membrane lipid determinants generally dictate the plasma membrane flux in eukaryotic cells. In this way, our results can for instance explain how environmental factors (e.g. oxygen supply) can indirectly influence endo- and exocytosis by intervening in lipid biosynthesis.
Acknowledgments D.D., B.C., I.B. and A.B. designed and performed the experiments. D.D., B.C., I.B., G.T. and A.B. discussed and interpreted the results and wrote the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to G.T. [TH558/28-1] and A.B. [BE1181/10-1]. All authors reviewed the final version of the manuscript and agreed with its content.
The volatile anaesthetic isoflurane decreases excitatory synaptic transmission primarily by presynaptic actions., , Isoflurane selectively inhibits action potential (AP)-evoked synaptic vesicle (SV) exocytosis from glutamatergic hippocampal neurones to a greater degree than from GABAergic neurones. However, the molecular basis for this differential inhibition and synaptic selectivity is unclear. Voltage-gated Ca channels (VGCCs) play a critical role in neurotransmission by mediating the Ca influx essential to triggering SV exocytosis., Because isoflurane does not alter the Ca-exocytosis coupling relationship in hippocampal neurones, its presynaptic selectivity must reside upstream of Ca influx; possible mechanisms include effects on voltage-gated Na channels, K channels, and VGCCs. Neurotransmitter phenotype-specific differences in expression, or anaesthetic sensitivity of presynaptic VGCC subtypes, or both could contribute to the observed selective inhibition of glutamatergic GABAergic SV exocytosis by isoflurane. We used live-cell fluorescence microscopy to examine the isoflurane sensitivity of P/Q-type and N-type VGCCs, which are the principal subtypes coupled to SV exocytosis in the central nervous system, at individual hippocampal synapses., Previous studies examining the effects of isoflurane on VGCCs have led to discordant results., , , , We tested the hypothesis that differential inhibition of specific presynaptic VGCC subtypes underlies the neurotransmitter phenotype selectivity observed for isoflurane inhibition of SV exocytosis.