Purification and assay of mitotic motors

Abstract

To understand how mitotic kinesins contribute to the assembly and function of the mitotic spindle, we need to purify these motors and analyze their biochemical and ultrastructural properties. Here we briefly review our use of microtubule (MT) affinity and biochemical fractionation to obtain information about the oligomeric state of native mitotic kinesin holoenzymes from eggs and early embryos. We then detail the methods we use to purify full length recombinant Drosophila embryo mitotic kinesins, using the baculovirus expression system, in sufficient yields for detailed in vitro assays. These two approaches provide complementary biochemical information on the basic properties of these key mitotic proteins, and permit assays of critical motor activities, such as MT-MT crosslinking and sliding, that are not revealed by assaying motor domain subfragments.

Figure. 1. Pan-kinesin antibody/MT affinity screen for native mitotic kinesins

(A) General strategy for purification of kinesin holoenzymes from native tissues. Kinesins are co-precipitated with MTs, and subsequently eluted from the MT-pellet by ATP. The resulting MAPs are further separated by conventional fractionation methods. (B) Detection of kinesin polypeptides that copurify with sea urchin egg microtubule precipitates by immunoblotting. Microtubules were prepared from AMPPNP (lane 1) or ATP (lane 2) treated cytosol. The microtubule proteins were analyzed on SDS-PAGE (left-hand panel) or on immunoblots probed with pan-kinesin antibodies (panel 2) that react with multiple kinesins. Using kinesin family-specific antibodies these were identified as kinesin-1 (KHC; panel 3), kinesin-5 (KRP₁₇₀; panel 4), kinesin-6 (KRP_l10; panel 5), and heterotrimeric kinesin-2 (KRP₈₅ and KRP₉₅; panel 6). (C) Effects of antibody microinjection on mitosis in fertilized sea urchin eggs. Embryos were shown 2hr (pane 1) and 4hr (pane 2) after fertilization. In these cells, injection of antibodies to some kinesins (e.g. kinesin-5, -6 and -12) altered spindle morphogenesis and inhibited normal cell division (upper panels), whereas antibodies to others (e.g. kinesin-1 and -2) had no effect on cell division (lower panels). Bar, 25μm.

Figure 2. Purification of the kinesin-5, KLP61F from Drosophila embryos

(A) SDS PAGE of fractions from gel filtration of Drosophila MAPs (L = loaded MAPs). Numbers above indicate gel filtration fractions. 130 on the right indicates KLP61F, KHC stands for kinesin heavy chain. (B) Corresponding immunoblot probed with pan-kinesin antibody. 90 on the right indicates the 90 kDa subunit of Ncd. (C) Coomassie-stained gel of sucrose density gradient fractions. Arrowhead indicates the peak fraction of KLP61F, 17% and 8% indicate percentage of sucrose at top and bottom of gradient, and molecular weight markers are indicated on the left.

Figure 3. Generation of recombinant baculoviruses and gene expression using the BAC-to-BAC expression system

(A) General scheme for expression mitotic kinesins by bac-to-bac expression system. (B) PCR analysis to check the insertion of KLP61F into Bacmid DNA. PUC/M13 amplification primers are directed at sequences on either side of the transposition site within the lacZα -complementation region of the bacmid. The expected results from PCR are as follows: Bacmid alone, ~300bp; Bacmid transposed with pFastBacHT, ~2430bp; Bacmid transposed with pFastBacHT containing KLP61F, ~6.0kb (2430bp+3502bp). (C) Western-blot screen of Sf9 cells transfected with baculovirus bearing KLP61F gene. Cell lysates were subjected to 7.5% SDS-PAGE. After protein bands were transferred onto a nitrocellulose membrane, KLP61F was detected by immunoblotting with mouse monoclonal anti-polyhistidine antibody(1:2000), and a secondary rabbit anti-mouse antibody (1:5000). Lane 1-13, cell lysates from 13 white colonies. Blue, cell lysate from blue colony.

Figure 4. Purification of Recombinant KLP61F and Ncd from the Baculovirus Expression System

(A-D) Commassie-blue-stained SDS-polyacrylamide gels show typical recombinant-motor-protein fractions obtained from the infected Sf9 cells. (A) Purification of rKLP61F. The following are shown: lane 1, Sf9 cell high-speed supernatant; lane 2, Ni-NTA affinity-column eluate; and lane 3, gel-filtration (Biogel A-15M, Biorad) fractionated and concentrated rKLP61F. (B) Purification of rNcd. The following are shown: lane 1, Sf9 cell high-speed supernatant; lane 2, Ni-NTA affinity-column eluate; and lane 3, gel-filtration fractionated and concentrated Ncd. (C) Purification of rKLP10A ; 1, Sf9 cell high-speed supernatant; lane 2, Ni-NTA affinity-column eluate; and lane3, gel-filtration (Superose 6 FPLC, GE Pharmacia) column eluate. (D) Purification of rPAV-KLP. The following are shown: lane 1, Sf9 cell high-speed supernatant; lane 2, Ni-NTA affinity-column eluate; and lane3, gel-filtration (Superose 6 FPLC, GE Pharmacia) column eluate. (E,F) Purification of KLP61F from Ni-NTA eluate by Bio-gel A15m Gel filtration. (E) Commassie blue-stained SDS-polyacrylamide gel shows the composition of protein fractions obtained during the purification of KLP61F from the gel-filtration chormotography. Void volume occurs at fractions 22-25 and included volume occurs at fractions 62-66 (F) Corresponding plot of A₂₈₀ versus fraction number shows one peak of monodisperse KLP61F.

Figure 5. Microtuble gliding assay (A, B) sliding assay (C, D) and bundling assay (E, F)

(A) Diagram of MT gliding assay. MTs are moved by motor proteins absorbed to the coverslip. (B) Fluorescence images of MTs moved by KLP61F immobilized on a coverslip. The scale bar represents 5μm. (C) Diagram of MT sliding assay. MTs attach via motor proteins to surface-immobilized MTs, and motors drive the MTs in solution to slide over the fixed MTs in an anti-parallel orientation. (D) Fluorescence images of one MT being slid by KLP61F over another fixed MT. The scale bar represents 5μm. (E) Diagram of Bundling Assay. Mtitotic motors crosslink adjacent MTs into bundles. (F) Purified rKLP61F, rNcd, and rPav-KLP can bundle MTs in 1mMATP. Fluorescence microscopy shows that purified rKLP61F, rNcd and rPav-KLP have obvious bundling activity. In the presence of 1 mM ATP, free MTs cannot be bundled by HK560 but can form robust MT bundles in the presence of rKLP61F, rNcd, and rPav-KLP. The salt condition for HK560, KLP61F and Ncd is 75 mM KCl, while for Pav-KLP is 200 mM KCl. The scale bar represents 10 μm.