The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes

Abstract

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Figure 1

Formation of a tubular network in crude cytoplasmic extracts of Xenopus oocytes. Cytoplasmic extract was diluted 1:4 with acetate buffer containing 2 mM ATP and placed in a microscope flow chamber. The same area was monitored over a period of 60 min, and photos were taken at intervals from the video recording. After 2 to 5 min, microtubules became visible at the surface of the cover glass (A) and some membrane tubules appeared about 10 min after incubation (B). An evenly spread membrane network was already established after about 20 min (C). (D–F) The density of the membrane networks increased and underwent constant rearrangements. Bar, 10 μm.

Figure 2

Effect of anti-dynein antibodies on the formation of membrane networks in cytoplasmic extracts of Xenopus oocytes. VEC-DIC images of cytoplasmic extracts supplemented 0.5 mg/ml antibody after an incubation for 60 min in a microscope flow chamber. (A and A′) Monoclonal antibody LEP100 specific for a lysosomal protein. (B and B′) Monoclonal antibody m74–1 specific for the 74-kDa dynein IC. (C and C′) Monoclonal antibody m74–2 also specific for the 74-kDa dynein IC. (A) A normal formation of polygonal network could be observed in control experiments, but the samples containing either of the monoclonal anti-dyneins (B and C) lacked any tubular networks. The anti-dyneins did not effect the polymerization of microtubules and their attachment to the cover glass. Bars: C, 10 μm; C′, 5 μm.

Figure 3

Determination of minimal concentration of antibody m74–2 for blocking the formation of tubular networks. VEC-DIC images of cytoplasmic extract supplemented with 125 μg/ml (A), 62.5 μg/ml (B), 31.5 μg/ml (C), 15.5 μg/ml (D), 7.5 μg/ml (E), and 0 μg/ml (F) monoclonal antibody m74–2. Tubular network formation was completely blocked at an antibody concentration of 15.5 μg/ml (D). Short membrane tubules were occasionally visible at an antibody concentration of 7.5 μg/ml (E), and a fully formed network was present in the control samples (F).

Figure 4

Dissociation of cytoplasmic dynein from membranes of cytoplasmic extracts of Xenopus oocytes analyzed by SDS-PAGE immunoblot. (A) Undiluted cytoplasmic extracts of interphase oocytes were supplemented with 0.5 mg/ml monoclonal antibody and incubated on ice for 3 h before resuspending the extract into 58% sucrose acetate buffer. The membrane fractions were isolated by flotation and collected at the 15–50% sucrose interphase. Lane 1, membrane fraction stained with Coomassie; lanes 2–5, immunoblot of membrane fractions stained with dynein IC-specific monoclonal antibody m74–2. Lane 2, membrane fractions of untreated cytoplasmic extract; lane 3, membrane fractions of cytoplasmic extract treated with 0.5 mg/ml m74–2; lane 4, membrane fraction of cytoplasmic extract treated with 0.5 mg/ml m74–1; lane 5, membrane fraction of cytoplasmic extract treated with 0.5 mg/ml control antibody LEP100. Molecular weight markers at left are as follows from top to bottom: 205,000, 97,000, 66,000, and 45,000. (B) Isolated membranes of Xenopus extracts were incubated with antibodies for 3 h on ice and then isolated by centrifugation through a 20% sucrose cushion. Lanes 1 and 1′, membrane fraction treated with 0.5 mg/ml control antibody; lanes 2 and 2′, membrane fraction treated with 0.5 mg/ml m74–2; lanes 1 and 2, stained with dynein IC-specific antibody m74–2; lane 1′ and 2′, stained with monoclonal antibody m150–1 specific for dynactin p150^Glued. (C) Immunoblot of antibody-treated membrane fractions from Xenopus extracts stained with polyclonal antibody (polydynH) raised against bacterially expressed dynein heavy chain from Dictyostelium (Vaisberg et al., 1993), dynactin p150^Glued-specific monoclonal antibody m150–1, dynein IC-specific monoclonal antibody m74–1, and dynein IC specific monoclonal antibody 70.1 (Steuer et al., 1990). Lane 1, microsomes isolated from bovine brain were used to demonstrate the specificity of the antibodies; lane 2, membrane fraction of cytoplasmic extract from Xenopus oocytes treated with control antibody; lane 3, membrane fraction of cytoplasmic extract of Xenopus oocytes treated with 0.5 mg/ml m74–2.

Figure 5

Monoclonal antibodies 74-1 and 74-2 block dynein IC–dynactin interaction in vitro. Rat brain cytosol (lane 1) was loaded onto a dynein IC affinity column (see MATERIALS AND METHODS) that was pretreated with BSA (lanes 2–4), m74–1 (lanes 5–7), or m74–2 (lanes 8–10). The columns were extensively washed and eluted with 1 M NaCl. The fractions were resolved by SDS-PAGE followed by transfer onto Immobilon and stained with Coomassie brilliant blue (A) and subsequently probed with antibodies to p150^Glued and centractin (B). Lane 1, cytosol; lanes 2, 5, and 8, flow-through fractions; lanes 3, 6, and 9, final wash fractions; lanes 4, 7, and 10, fractions eluted with 1 M NaCl.

Figure 6

Identification of the binding site of monoclonal antibodies m74–1 and m74–2 on dynein IC. (A) Immunoblot of truncation mutants of expressed dynein IC labeled with monoclonal antibody m74–1 and m74–2 or polyclonal antibody pan-IC specific for the whole dynein IC polypeptide. (B) Line diagram depicting dynein IC fragments used for mapping.