Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis

Abstract

Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast twohybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.

Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

“…The size of these early endosomes (approximately 1 mm) suggest that they may be clathrin-coated vesicles. 22,31 The absence of Sytox Green fluorescence in the nucleus of these cells confirmed that they were alive, so that the internalization of the FM dye cannot be attributed to cell death. In fact, cells pretreated with DMSO 10% died and serve as a positive control for Sytox Green stain (Fig.…”

Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis

“…The aggregation of PIN2:GFP at certain sites of the PM is similar to the behavior of DRP1A and 1C under the Tyrphostin A23 treatment. Dynamics of these proteins was disturbed and the size of the foci at the PM to which fluorescent protein-tagged DRP1s localize was increased (Konopka et al, 2008;Fujimoto et al, 2010). Moreover, the localization pattern of PIN2:GFP in the ap2m-1 mutant filaments was phenocopied by treatment of the wild type with Tyrphostin A23.…”

CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication

“…4c,d), indicating that continuous vesicle trafficking is required for creating CHER1 polarization in SE. Furthermore, we analysed the behaviour of two dynamin-related proteins, DRP1A and DRP1C, which are involved in the clathrinmediated endocytosis in plants [42][43][44] . As previously reported, both DRP1A and DRP1C localize to the cell plate and the plasma membrane of expanding and fully expanded interphase cells 44,45 .…”