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Visualizing Endocytosis by Variable Angle Epifluoresence Microscopy (VAEM)

Clathrin-mediated endocytosis (CME) plays a number of critical roles in plant development.  These include retrieval of excess membrane material during tip-directed growth and cell plate maturation as well as roles in the signalling of many plant hormones.  For example, CME is required for the localization and signaling of receptors for brassinosteroids and the maintenance of the polar localization of auxin transporters necessary for establishment of auxin gradients.

Despite its developmental importance, the study of endocytosis mechanisms in plants is in its infancy, and most of what is known is by inference from endocytosis in yeast and mammals.

Image of endocytosis

The core endocytic machinery, including clathrin, the AP-2 complex, and some clathrin accessory proteins, appears to be conserved between plants and animals.  However, there are also some significant differences, such as the involvement of two distinct families of dynamins (DRP1 and DRP2) and the absence of some accessory proteins such as endophilin and amphiphysin.  Also, it is unclear what role, if any, PIP(4,5)P2 and F-actin play in endocytosis in plants.

CLC2-GFP localizes to the plasma membrane (cell outlines), the trans-Golgi network (bright dots) and forming cell plates (bright lines) in Arabidopsis rootsCLC2-GFP localizes to the plasma membrane (cell outlines), the trans-Golgi network (bright dots) and forming cell plates (bright lines)  in Arabidopsis roots - all expected sites of clathin-mediated trafficking.

One tool that has been used extensively for the study of clathrin-mediated endocytosis in mammalian cells is Total Internal Reflection Fluorescence Microscopy (TIRFM). It is not clear to what extent TIRFM can be used in plants because of the presence of a thick cell wall. However, similar results can be obtained using a related microscopy technique termed Variable-Angle Epifluoresence Microscopy (VAEM).

VAEM diagram

Like TIRFM, VAEM allows high resolution visualization of fluorescently labeled endocytic structures at the cell cortex. It allows faster imaging than confocal microscopy, and is better for imaging three-dimensional particles (such as clathrin-coated structures) because it is not limited to a narrow focal plane. As compared to traditional epifluorescent microscopy, it gives much clearer images of structures at the cell cortex by eliminating signals from the rest of the cell. This is particularly important when imaging fluorophores attached to peripheral membrane proteins such as dynamins that have a significant cytoplasmic background. 

photo of a root expressing DRP1C-GFP viewed by standard or variable angle epifluoresence. A root expressing DRP1C-GFP viewed by standard or variable angle epifluoresence.

Time-lapse imaging by VAEM can be technically challenging because any vertical motion of the tissue being imaged necessitates constant adjustment of the focus.  However, once mastered it can provide a wealth of information on endocytosis or other processes at the cell cortex. Furthermore, simultaneous dual-color imaging allows the examination of spatial and temporal colocalization between two separate endocytic proteins.   For more information on VAEM and its uses, see Konopka and Bednarek (2007) Plant J. 53, 186-196

We have used VAEM to show that the dynamin-related proteins DRP1A and DRP1C are involved in clathrin-mediated endocytosis, and to define the dynamics and drug sensitivities of clathrin-coated particles (see our research page).   Other groups have used VAEM in a similar manner to look at the role of DRP2 in endocytosis (Fujimoto et al. 2010 PNAS 107, 6094).  Below are some images and movies of different endocytic markers visualized by VAEM.

Images and movies of different endocytic markers visualized by VAEM

Movie of CLC2-GFP | Movie of DRP1C-GFP | Movie of DRP1C-GFP CLC-mOrange

All movies play at 2x real speed.  All movies are from Konopka et al. 2008 Plant Cell 20, 1363.