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. 2001 Nov;13(11):2427-39.
doi: 10.1105/tpc.010225.

A novel plant kinesin-related protein specifically associates with the phragmoplast organelles

Y R Lee  1 , H M Giang, B Liu
Affiliations

A novel plant kinesin-related protein specifically associates with the phragmoplast organelles

Y R Lee et al. Plant Cell. 2001 Nov.

Abstract

In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the phragmoplast.

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Figures

Figure 1.
Figure 1.
AtPARKP2 Is a Divergent KRP. (A) The amino acid sequence was deduced from AtPAKRP2 cDNA clones. The N-terminal region similar to KRP motor domains is shaded. The highly conserved peptides in the motor domain are highlighted with black background. (B) Phylogenetic tree built from a sequence alignment of KRP motor domains.
Figure 2.
Figure 2.
AtPAKRP2 Structure and Its ATP-Dependent MT Binding Activity. (A) Coiled-coil domains of AtPAKRP2 were predicted by the Lupus algorithm. The x axis represents the amino acid residue number, and the y axis represents the probability of the formation of coiled coils. A P value >0.5 indicated that the region likely forms a coiled coil. (B) Schemes of AtPARKP2 and its protein constructs used for MT binding and antibody preparation. The relative positions of the motor, coiled coil, and tail are indicated. (C) The MT binding property of the GST-AtPAKRP2-M fusion protein was demonstrated by cosedimentation with MTs. The resulting supernatant (S) and pellet (P) were analyzed by SDS-PAGE and visualized by Coomassie blue staining. (−), the GST-AtPARKP2-M fusion protein without addition of MTs; MTs, the fusion protein incubated with MTs alone; MTs + ATP, the fusion protein incubated with MTs and 2 μM ATP; MTs + AMPPNP, the fusion protein incubated with MTs and 2 μM AMPPNP.
Figure 3.
Figure 3.
Immunodetection of the AtPAKRP2 Protein in Arabidopsis. (A) Immunoblots containing proteins extracted from inflorescence buds were stained with affinity-purified anti-AtPAKRP2-N′ antibodies (lane 1) or anti-AtPAKRP2-N′ antibodies incubated with GST-AtPAKRP2-N′ proteins before immunoblot staining (lane 2). Molecular mass markers (kD) are shown at left. (B) Intracellular localization of AtPAKRP2 in an Arabidopsis root tip cell at cytokinesis. AtPAKRP2 is shown in green, and DNA is shown in blue. (C) Anti–AtPAKRP2-N′ staining was absent in an Arabidopsis root tip cell at telophase when the antibodies were incubated with the GST-AtPAKRP2-N′ protein before immunofluorescence staining. (D) MTs (red) and DNA (blue) in the same cell as in (C). Bar = 5 μm for (B) to (D).
Figure 4.
Figure 4.
AtPAKRP2 Was Localized among Interzonal MTs. Triple localizations of AtPAKRP2 with anti-AtPAKRP2-N′ ([A], [E], and [I]), of MTs with anti–α-tubulin ([B], [F], and [J]), and of DNA with DAPI ([C], [G], and [K]) are shown in Arabidopsis root tip cells at interphase ([A] to [D]), metaphase ([E] to [H]), and late anaphase ([I] to [L]). The pseudocolored composite images ([D], [H], and [L]) show AtPAKRP2 in green, α-tubulin in red, and DNA in blue. Bar in (L) = 5 μm for (A) to (L).
Figure 5.
Figure 5.
Association of AtPAKRP2 with the Phragmoplast. Triple localizations of AtPAKRP2 with anti-AtPAKRP2-N′ ([A], [E], and [I]), of MTs with anti-α-tubulin ([B], [F], and [J]), and of DNA with DAPI ([C], [G], and [K]) are shown in Arabidopsis cells during telophase/cytokinesis. Composite images ([D], [H], and [L]) are pseudocolored with AtPAKRP2 shown in green, α-tubulin shown in red, and DNA shown in blue. Bar in (L) = 5 μm for (A) to (L).
Figure 6.
Figure 6.
Requirement of MTs for AtPAKRP2 Localization. (A) After APM treatment, AtPAKRP2 appeared in a diffuse pattern in the cytoplasm in an Arabidopsis root tip cell after the completion of mitosis. (B) MTs were depolymerized completely in this cell, as indicated by anti–α-tubulin. (C) Daughter nuclei stained with DAPI. (D) to (F) A control root tip cell at a similar stage as in the cell (A) to (C), (shown by DAPI staining of the nuclei in [F]) was treated with isopropanol alone. AtPAKRP2 localization was not disturbed (D), nor were phragmoplast MTs (E). Bar in (F) = 5 μm for (A) to (F).
Figure 7.
Figure 7.
Distinct Localization Patterns of AtPAKRP2 and AtPAKRP1. Arabidopsis root tip cells at early cytokinesis ([A] to [C]) and late cytokinesis ([D] to [F]) were stained with rat anti–AtPAKRP2-T ([A] and [D]) and rabbit anti–AtPAKRP1-C ([B] and [E]) antibodies. Daughter nuclei are shown by DAPI staining ([C] and [F]). AtPAKRP1 appeared much more discretely at the equatorial plane than did AtPAKRP2. Bar in (F) = 5 μm for (A) to (F).
Figure 8.
Figure 8.
Association of AtPAKRP2 with the Insoluble Cytosolic Fraction and Disturbance of AtPAKRP2 Localization by BFA. (A) Inflorescence buds of Arabidopsis were used for subcellular fractionation. Equal amounts of protein from total protein extract (lane 1), the cytosolic fraction (lane 2), and the insoluble cytosolic fraction (lane 3) were subjected to immunoblotting with anti–AtPAKRP2-N′ antibodies. The arrow indicates a single band recognized by the anti–AtPAKRP2-N′ antibodies, which is enriched in the soluble cytosolic fraction. (B) to (D) A tobacco BY-2 cell was treated with BFA in methanol. (E) to (G) A tobacco BY-2 cell was treated with methanol alone. (H) to (J) A tobacco BY-2 cell was fixed and stained without any treatment. The cells were double stained with anti–AtPAKRP2-N′ ([B], [E], and [H]) and anti–α-tubulin ([C], [F], and [I]) antibodies. (D), (G), and (J) show relative intensities of immunofluorescence of AtPAKRP2 (thick gray lines) and tubulin (thin black lines). The x axis represents the position of the data point, which was aligned to the micrographs, and the y axis represents the relative fluorescence intensity with the value given arbitrarily by ImagePro software. Bar in (I) = 10 μm for (B) to (I).
Figure 9.
Figure 9.
Distinct Localization Patterns of AtPAKRP2 and KNOLLE in the Phragmoplast. Triple localizations were performed with anti–AtPAKRP2-T (A), anti-KNOLLE (B), and DAPI (C) in B. oleracea cells undergoing cytokinesis as revealed by the appearance of the daughter nuclei (C). The composite image (D) was pseudocolored, with AtPAKRP2 shown in green, KNOLLE shown in red, and DNA shown in blue. Bar in (D) = 5 μm for (A) to (D).
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