Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells

Abstract

We have isolated a myosin (referred to as 170-kD myosin) from lily pollen tubes, which consists of 170-kD heavy chain and calmodulin (CaM) light chain and is responsible for cytoplasmic streaming. A 170-kD polypeptide that has similar antigenicity to the 170-kD myosin heavy chain of lily pollen tubes was also present in cultured tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells, and possessed the ability to interact with F-actin in an ATP-dependent manner. In addition to this myosin, we identified biochemically another kind of myosin in BY-2 cells. This myosin consisted of a CaM light chain and a 175-kD heavy chain with antigenicity different from the 170-kD myosin heavy chain. In the present study, we referred to this myosin as 175-kD myosin. This myosin was able to translocate rhodamine-phalloidin (RP)-labeled F-actin at an average velocity of about 9 &mgr;m/s in the motility assay in vitro. In contrast, the sliding velocity of RP-labeled F-actin translocated by fractions containing the 170-kD myosin was 3 to 4 &mgr;m/s. The velocity of cytoplasmic streaming in living BY-2 cells ranged from 2 to 9 &mgr;m/s. The motile activity of 175-kD myosin in vitro was inhibited by Ca(2+) at concentrations higher than 10(-6) M. Immunoblot analyses using an antiserum against the heavy chain of 170- or 175-kD myosin revealed that in tobacco plants, the 175-kD myosin was expressed in leaf, stem, and root, but not in germinating pollen, while 170-kD myosin was present in all of these plant parts and in germinating pollen. These results suggest that the two types of myosins, 170 and 175 kD, presumably participate in cytoplasmic streaming in BY-2 cells and other somatic cells of tobacco plants.

Figure 1

Identification of 170-kD myosin in BY-2 cells. A, Immunoblotting of a crude protein sample from BY-2 cells. Lane a, Coomassie Brilliant Blue staining of a 6% (w/v) SDS-polyacrylamide gel; lane b, immunoblotting using AS-170. The arrowheads 1 and 2 indicate the 170- and 165-kD polypeptides, respectively. B and C, Coomassie Brilliant Blue staining of a 6% (w/v) SDS-polyacrylamide gel of representative fractions from co-precipitation steps with F-actin and immunoblotting of the same fractions with AS-170, respectively. Co-precipitant with F-actin (lane a) after centrifugation of a mixture of a crude cell extract and F-actin. Supernatant (lane b) and pellet (lane c) after the co-precipitant shown in lane a was treated with EMP solution. Supernatant (lane d) and pellet (lane e) after ATP extraction of the pellet shown in lane c. The arrows 1 to 8 indicate polypeptides that appear to be dissociated from F-actin in an ATP-dependent manner. The arrowheads 1 and 2 indicate the 170- and 165-kD polypeptide, respectively. Arrows a to c indicate proteolytic fragments of the 170-kD myosin heavy chain or 165-kD polypeptide (see the text). The molecular masses (×10³) of standard proteins are indicated on the left of A in kilodaltons.

Figure 2

Hydroxylapatite column chromatography of the ATP extract. A, Silver staining of a 6% (w/v) SDS-polyacrylamide gel. B, Immunoblotting of the same fractions using AS-170. Numbers on the top of gels indicate fraction numbers. After the application of the ATP extract shown in Figure 1B on a hydroxylapatite column, the adsorbed materials were eluted with a discontinuous gradient of 5 mm (fraction 1–5), 150 mm (fraction 6–11), and 300 mm (fraction 12–17) potassium phosphate buffer. The arrowheads and arrow indicate 170-kD myosin and the 120-kD degradation product of the 170-kD heavy chain, respectively.

Figure 3

Motile activity of hydroxylapatite column fractions shown in Figure 2. A, Percentage of translocated F-actin. B, Sliding velocity.

Figure 4

DE-52 ion-exchange column chromatography of the hydroxylapatite column fractions. Fractions 2 and 3 shown in Figure 2 were applied to a DE-52 column and the adsorbed materials were eluted with a linear gradient of 5 to 300 mm KCl. A 6% (w/v) SDS-polyacrylamide gel was stained with silver. Fraction numbers are shown on the top of the figure.

Figure 5

Motile activity of each DE-52 column fraction shown in Figure 4. A, Percentage of translocated F-actin. B, Sliding velocity.

Figure 6

Co-precipitation experiment of polypeptides in DE-52 column fractions with F-actin. A, Silver staining of a 6% (w/v) SDS-polyacrylamide gel. B, Silver staining of a 15% (w/v) SDS-polyacrylamide gel. C, Immunoblotting of the same samples shown in B with AS-CaM. Fractions 9 to 15 shown in Figure 4 were pooled (lanes a) and mixed with F-actin (lanes b). Supernatant (lanes c) and pellet (lanes d) after centrifugation of the mixture shown in lanes b. Supernatant (lanes e) and pellet (lanes f) after ATP treatment of the pellet shown in lanes d. The arrowhead and arrow indicate the 175-kD polypeptide and the 18-kD band, respectively. The percentage of translocated F-actin in motile assay was 79%, 2%, and 92% for fractions a, c, and e, respectively.

Figure 7

The effect of Ca²⁺ on the motile activity of the 300 mm potassium phosphate eluate corresponding to fraction 13 shown in Figure 2 (A) and isolated 175-kD myosin (B). Black bars and white bars represent the percentage of translocated F-actin and sliding velocity, respectively.

Figure 8

Cross-reactivity of antibodies against the 175-kD myosin heavy chain. A, Immunoblotting of isolated 175-kD myosin shown in Figure 6. Lane a, Coomassie Brilliant Blue staining of a 6% (w/v) SDS-polyacrylamide gel; lane b, immunoblotting using AS-175; lane c, immunoblotting using AS-170. B, Immunoblotting of the ATP extract of the co-precipitant with F-actin in a crude cell extract. Lane a, Silver staining of a 6% (w/v) SDS-polyacrylamide gel; lane b, immunoblotting using AS-175; lane c, immunoblotting using AS-170. C, Immunoblotting of a crude protein sample from BY-2 cells. Lane a, Coomassie Brilliant Blue staining of a 6% (w/v) SDS-polyacrylamide gel; lane b, immunoblotting using AS-175; lane c, immunoblotting using non-immune serum; lane d, immunoblotting using AAB-175. The arrowheads 1 and 2 indicate the 175- and the 170-kD myosin heavy chains, respectively. The arrows a and b indicate the 135- and the 120-kD degradation product of the 170-kD myosin heavy chain, respectively. The region marked by a bracket shows the low-molecular-mass polypeptides reacting with AS-175.

Figure 9

Immunoblotting of a crude protein sample from leaf (A), root (B), stem (C), and germinating pollen (D) of tobacco plants. Lanes a, Coomassie Brilliant Blue staining of a 6% (w/v) SDS-polyacrylamide gel; lanes b, immunoblotting using AS-170; lanes c, immunoblotting using AS-175. A total of 100, 30, 22, and 155 μg of protein from leaf, root, stem, and germinating pollen, respectively, were applied to SDS gels. The arrows and the arrowheads indicate the 170- and the 175-kD bands, respectively. The region marked by a bracket shows the low-molecular-mass polypeptides reacting with AS-175.