Mechanisms underlying coordinated regulation of exo- and endocytosis in BY-2 cells
Final Report Abstract
Over the past years exo- and endocytosis have been recognized as an essential part of many physiological processes in plant cells ranging from cell division to signalling and development. In order to understand normal function and pathological conditions of plant organisms a knowledge of the rather complex processes of vesicle fusion and fission is therefore essential. To analyse the kinetic and size of single exo- and endocytotic events in BY-2 protoplasts we employed cell-attached membrane capacitance measurements which allow us to follow fusion and fission of single vesicles in real time in single living cells. These measurements revealed different kinetic modes of fusion and fission of single vesicles which could be grouped into four categories according to their different kinetics: (i) transient fusion of exocytotic vesicles, (ii) transient fission of an endocytotic vesicle, (iii) permanent fusion, and (iv) permanent fission. This study established BY-2 protoplasts as a model system of exo- and endocytosis by membrane capacitance measurements. Building on these results our further studies focused on mechanisms of endocytosis in BY-2 cells. In eukaryotic cells several pathways for the internalisation of plasma membrane proteins and extracellular cargo molecules exist. These endocytic pathways can be grouped into clathrin-dependent and clathrin-independent endocytosis. While the former one has been described to be involved in a variety of cellular processes in plants, the latter one has so far only been identified in animal and yeast cells. Using fluorescent imaging studies in addition to membrane capacitance measurements we identified a clathrin-independent endocytic pathway which is involved in the uptake of glucose into BY-2 cells. Membrane capacitance measurements furthermore revealed the stimulation of endocytic activity by extracellular glucose. These studies therefore provide novel insights into the mechanism of endocytosis in plants. We also tested a new marker for the investigation of endocytosis in turgescent cells and protoplasts. To investigate clathrin-independent pathways the generally used endocytic membrane marker FM4-64 cannot be employed because endocytosis of FM4-64 has been shown to strongly depend on clathrin. We therefore studied the application fluorescent nano beads to follow endocytosis in BY-2 cells and protoplasts. The results demonstrated that small nano beads provide a useful tool to follow endocytosis in plant cells. Inhibitor studies implied that uptake of nano beads involves both, clathrin-dependent and clathrin-independent endocytosis. This points to a role of at least two endocytic mechanisms in the constitutive uptake of membrane and fluid in plant cells.
Publications
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(2011) Modes of exocytotic and endocytotic events in tobacco BY-2 protoplasts. Molecular Plant 4: 241-251
Bandmann V, Kreft M, Homann U
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(2012) Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts. The Plant Journal 70: 578–584
Bandmann V, Homann U
