4. Dynamin-related proteins

Fig 1. Phragmoplastin, the first protein marker of the cell plate.

(A-D) A cytokinetic tobacco BY-2 cell was stained with DAPI for nuclear DNA (A), anti-tubulin monoclonal antibody for phragmoplast microtubules (B) or anti-soybean phragmoplastin polyclonal antibodies (C). The fluorescence signals were recorded separately in black-and-white photographs. Artificial colors are assigned to DAPI staining (in blue), microtubules (in red) and cell plate (in green). The three photographs are superimposed to indicate the relative subcellular localization of phragmoplastin (D; Gu and Verma 1996). N, nuclear DNA. M, phragmoplast microtubules. CP, cell plate. (E). Green fluorescent protein (GFP)-tagged phragmoplastin expressed in a tobacco BY-2 cell (Gu and Verma 1997). 

Fig 2. DRP1 and DRP2 act as tubulase and pinchase that control exocytosis and endocytosis, respectively.

(A-D) Vesicle tubulation and membrane vesiculation are mediated by structurally related DRP1 (phragmoplastins) and DRP2 (dynamins). (A,C) DRP1 acts as a tubulase and forms highly ordered spiral polymers on the surface of vesicles. Upon GTP hydrolysis, DRP1 provides mechanical force to convert vesicles to tubular structures. (B,D) DRP2, a pinchase, forms a collar ring surrounding the junction region between a budding endocytic vesicle and plasma membrane. (E) Electron micrograph showing a long dumbbell-shaped tubule that is coated with DRP1 tubulase at the nascent cell plate of tobacco BY-2 cells (Samuels et al. 1995). (F) Electron micrograph showing the pinching off of endocytic vesicles at the plasma membrane that are formed by the defective dynamin mutant shibire (Koenig and Ikeda 1989).

Fig 3. Classification and functional domains of DRPs in Arabidopsis.

There are 16 genes encoding dynamin-related proteins (DRPs) in the Arabidopsis genome. They are grouped into five functional families and one unknown group. DRP1 act as tubulase to convert vesicles into membranous tubules. DRP2 functions as pinchase to create vesicle from the plasma membrane. DRP3 and DRP5 participate in mitochondrial and chloroplast division, respectively. DRP4 members are involved in anti-virus processes.

The percentage of amino acid identity with DRP1A is shown after each peptide. The identity (%) between the two adjacent peptides is shown at the left. DYN1, dynamin GTPase domain which includes a GTP-binding motif (GXXXSGKS/T) and a dynamin signature. FBD, FeoB domain which is found in the ferrous iron transport protein B.  DYN2, dynamin central region. PH, pleckstrin homology domain which binds to membrane phospholipids such as PI-3-P and PI-4-P. GED, dynamin GTPase effector domain. PRD, proline-rich domain which interacts with SH3-domain proteins. 

Figure 4. Subcellular localization of GFP-tagged DRP1A, DRP1C and DRP1E in BY-2 cells. 

(A-C) Association of DRP1A with microtubules and the forming cell plate. (A) In the G1 phase cell, DRP1A is associated with the strand-like structures distributed only in the cortex region of the cell. (B) In the S phase cell, the nucleus (N) is centralized and DRP1A is distributed on strand-like structures that initiate from the perinuclear region and extend along the cytoplasmic strands towards the cell cortex. (C) In the metaphase cell, DRP1A is found on the spindles (SPD). From late metaphase to early anaphase, DRP1A is associated with the phragmoplast including the two sets of microtubule arrays and the forming cell plate (CP).

(D-F). Association of DRP1C with microtubules and the forming cell plate. (D) DRP1C is associated with the cortical microtubules in the G1 cell and with the mitotic spindle (SPD) in the mid phase cell. (E) DRP1C is largely targeted to the forming cell plate (CP) at anaphase. When the 3-D confocal image in E is turned -300 around the X-axis and the phragmoplast region is amplified (F), DRP1C is largely located on the edges of the cell plate, forming a ring-like structure.

(G-I). Localization of GFP-DRP1E fusion protein in transgenic BY-2 cells. (G) Side view of the cell plate (CP) of an anaphase cell. N, daughter nuclei. (H)  Front view of the cell plate (CP) of a similar cell as in A. (I) Confocal image of DRP1E localization. Note that it is localized to the disk-like structure of an early cell plate filled with the tubular-network.