Carrillo, Lucia (2015)
Application of high-resolution membrane capacitance measurements in the study of exocytosis and endocytosis in Saccharomyces cerevisiae.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung
Kurzbeschreibung (Abstract)
High resolution cell-attached patch-clamp recordings are used to analyze the kinetics of exo- and endocytosis in yeast. This technique is based on the fact that physical properties of cell membranes are comparable to the properties of a RC-circuit, consisting of a resistor and a capacitor in parallel. This means that changes in the plasma membrane surface, resulting from fusion and fission of vesicles, can be measured as changes in the membrane capacitance. Application of cell-attached patch-clamp recordings to Saccharomyces cerevisiae facilitates detection of exo- and endocytotic events, as discrete changes in the membrane capacitance at high temporal and spatial resolution. The results show four different kinetic modes of exo- and endocytosis in yeast, which were already reported from other eukaryotes. These modes include transient and permanent fusion and fission of vesicles. The measured capacitance changes were predominantly in the range of 0.2 - 1 fF, which corresponds to vesicle diameters of 90 - 200 nm. The vesicle size distribution showed a median of about 132 nm for endocytotic and 155 nm for exocytotic vesicles. Under well-defined nutrient conditions endocytotic and exocytotic events occurred at frequencies of 8.1 and 12.3 events per hour, respectively. In comparison, protoplasts deprived of glucose showed lower frequencies with nearly four events/h for exocytotic, and two events/h for endocytotic events. This result indicates energy requirement for exo- and endocytosis in yeast. Via capacitance measurements it was feasible to examine exo- and endocytosis frequencies, to determine vesicle sizes and to detect fusion pores in yeast cells. Because of the latter it was also possible to establish a rough classification of fusion pores conductance and diameters. A temperature sensitive SEC mutant sec6-4 inhibited in the secretory pathway revealed a direct relationship between recorded capacitance changes in the plasma membrane and exocytotic events. In this context cells of the aforementioned mutant were incubated at the restrictive temperature of 37 °C resulting in an accumulation of vesicles in the cytosol. After incubation 71.6 exocytotic and 16 endocytotic events/h were measured. As a control sample, cells were incubated at the permissive temperature of 25 °C. The measured frequencies were comparable to the reference strain BY4741, which results in 15.1 events/h for exocytotic and 9.2 events/h for endocytotic events. This demonstrates that event frequencies can be modulated. Through this study it was possible to demonstrate that capacity measurements can be applied to yeast cells for the detection of single fission and fusion events. Analyses on yeast protoplasts suggest that this system can be used for the investigation of exo- and endocytosis via capacity measurements. This provides a new tool for analyzing both processes in yeast.
Typ des Eintrags: | Dissertation | ||||
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Erschienen: | 2015 | ||||
Autor(en): | Carrillo, Lucia | ||||
Art des Eintrags: | Erstveröffentlichung | ||||
Titel: | Application of high-resolution membrane capacitance measurements in the study of exocytosis and endocytosis in Saccharomyces cerevisiae | ||||
Sprache: | Englisch | ||||
Referenten: | Bertl, Prof. Dr. Adam ; Thiel, Prof. Dr. Gerhard | ||||
Publikationsjahr: | 2015 | ||||
Ort: | Darmstadt | ||||
Datum der mündlichen Prüfung: | 27 November 2015 | ||||
URL / URN: | http://tuprints.ulb.tu-darmstadt.de/5207 | ||||
Kurzbeschreibung (Abstract): | High resolution cell-attached patch-clamp recordings are used to analyze the kinetics of exo- and endocytosis in yeast. This technique is based on the fact that physical properties of cell membranes are comparable to the properties of a RC-circuit, consisting of a resistor and a capacitor in parallel. This means that changes in the plasma membrane surface, resulting from fusion and fission of vesicles, can be measured as changes in the membrane capacitance. Application of cell-attached patch-clamp recordings to Saccharomyces cerevisiae facilitates detection of exo- and endocytotic events, as discrete changes in the membrane capacitance at high temporal and spatial resolution. The results show four different kinetic modes of exo- and endocytosis in yeast, which were already reported from other eukaryotes. These modes include transient and permanent fusion and fission of vesicles. The measured capacitance changes were predominantly in the range of 0.2 - 1 fF, which corresponds to vesicle diameters of 90 - 200 nm. The vesicle size distribution showed a median of about 132 nm for endocytotic and 155 nm for exocytotic vesicles. Under well-defined nutrient conditions endocytotic and exocytotic events occurred at frequencies of 8.1 and 12.3 events per hour, respectively. In comparison, protoplasts deprived of glucose showed lower frequencies with nearly four events/h for exocytotic, and two events/h for endocytotic events. This result indicates energy requirement for exo- and endocytosis in yeast. Via capacitance measurements it was feasible to examine exo- and endocytosis frequencies, to determine vesicle sizes and to detect fusion pores in yeast cells. Because of the latter it was also possible to establish a rough classification of fusion pores conductance and diameters. A temperature sensitive SEC mutant sec6-4 inhibited in the secretory pathway revealed a direct relationship between recorded capacitance changes in the plasma membrane and exocytotic events. In this context cells of the aforementioned mutant were incubated at the restrictive temperature of 37 °C resulting in an accumulation of vesicles in the cytosol. After incubation 71.6 exocytotic and 16 endocytotic events/h were measured. As a control sample, cells were incubated at the permissive temperature of 25 °C. The measured frequencies were comparable to the reference strain BY4741, which results in 15.1 events/h for exocytotic and 9.2 events/h for endocytotic events. This demonstrates that event frequencies can be modulated. Through this study it was possible to demonstrate that capacity measurements can be applied to yeast cells for the detection of single fission and fusion events. Analyses on yeast protoplasts suggest that this system can be used for the investigation of exo- and endocytosis via capacity measurements. This provides a new tool for analyzing both processes in yeast. |
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URN: | urn:nbn:de:tuda-tuprints-52077 | ||||
Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften | ||||
Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Yeast Membrane Biology |
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Hinterlegungsdatum: | 20 Dez 2015 20:55 | ||||
Letzte Änderung: | 20 Dez 2015 20:55 | ||||
PPN: | |||||
Referenten: | Bertl, Prof. Dr. Adam ; Thiel, Prof. Dr. Gerhard | ||||
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 27 November 2015 | ||||
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