Hydrodynamic flow in the cytoplasm of plant cells.

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007) shows that active transport of organelles gives rise to a drag in the cytosol, setting up a hydrodynamic flow, which contributes to a fast distribution of proteins and nutrients in plant cells. Here, we show experimentally that actively transported organelles produce hydrodynamic flow that significantly contributes to the movement of the molecules in the cytosol. We have used fluorescence recovery after photobleaching and show that in tobacco Bright Yellow 2 (BY-2) suspension cells constitutively expressing cytoplasmic green fluorescent protein (GFP), free GFP molecules move faster in cells with active transport of organelles than in cells where this transport has been inhibited with the general myosin inhibitor BDM (2,3-butanedione monoxime). Furthermore, we show that the direction of the GFP movement in the cells with active transport is the same as that of the organelle movement and that the speed of the GFP in the cytosol is proportional to the speed of the organelle movement. In large BY-2 cells with fast cytoplasmic streaming, a GFP molecule reaches the other side of the cell approximately in the similar time frame (about 16 s) as in small BY-2 cells that have slow cytoplasmic streaming. With this, we suggest that hydrodynamic flow is important for efficient transport of cytosolic molecules in large cells. Hydrodynamic flow might also contribute to the movement of larger structures than molecules in the cytoplasm. We show that synthetic lipid (DOPG) vesicles and 'stealth' vesicles with PEG phospholipids moved in the cytoplasm.

Microinjecting FM4-64 validates it as a marker of the endocytic pathway in plants.

The amphiphilic dye FM4-64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4-64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4-64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4-64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4-64 interacts with.