1 C, right). For functional analysis, we began by constructing several plasmids to express wild-type and mutant PRC1 (Fig. suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in -helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, HAMNO and that distinct functions are associated with modular elements of the primary sequence. WT PRC1 and PRC1EE HAMNO both pellet in presence of MTs. M273 and C273 also pellet (P) in presence of MTs, whereas C439 and N305 remain mostly in the supernatant (S). The arrow indicates the position of tubulin. Open in a separate window Figure 1. The distribution of the endogenous PRC1 and its chimeras in HeLa cells. (A) The localization of the endogenous PRC1 and TD-60. (aCc) HeLa cells were labeled with the affinity-purified antibody against PRC1 (green) and with propidium iodide for DNA (red). PRC1 is present in nuclei in interphase cells (a), and then becomes localized to the spindle upon entry into mitosis (b), and concentrates in the spindle midzone during late anaphase (c). (dCf) Double labeling of PRC1 (green) and TD-60 (red) during mitosis. (d) In metaphase, TD-60 is bound to the kinetochores at the metaphase plate, whereas PRC1 is associated with the spindle, enriched on interdigitating MTs. In late mitosis, the two antigens largely colocalize at the cleavage furrow (e) and midbody (f). PRC1 appears restricted to the Flemming body. Bars, 10 m. (B) Schematic representation of the PRC1 chimeras. PRC1 was expressed in bacteria or HeLa cells as a tagged protein. For the first two constructs, the complete coding region of PRC1 (1C620 aa) was fused downstream to a 6-His tag (His-PRC1) or an EGFP tag (EGFPCPRC1). The last construct allowed the expression of a COOH terminus truncation of PRC1 fused upstream to the EGFP (PRC1CEGFP). Both types of EGFP chimeras were used to overexpress the PRC1 mutant proteins. The PRC1AA protein is a null phosphorylation mutant, with both Thr 470 and Thr 481 (red vertical bars) mutated into Ala. The PRC1EE protein, with both the Thr 470 and Thr 481 mutated into Glu, mimics phosphorylation. The blue boxes indicate the regions of PRC1 with a high probability of multicoil formation (residues 35C85, 89C135, 210C251, 384C411, and 437C464). (C) Expression of the endogenous PRC1 and chimeras in HeLa cells. Extracts from HeLa, either untreated or transfected with one of the PRC1 chimeras (EGFPCPRC1, PRC1CEGFP, and PRC1CEGFPAA), or with a control plasmid expressing only EGFP, were subjected to MCM7 electrophoresis on 8% polyacrylamide gels and immunoblotted with the anti-EGFP antibody (left) or anti-PRC1 antibody (right). (Right) The anti-PRC1 antibody recognizes the endogenous PRC1 (arrowhead) in all extracts along with a band at the expected molecular mass of the EGFPCPRC1 fusion protein (arrow). PRC1CEGFP and the mutant PRC1CEGFPAA fusions were not recognized because they lack the COOH- terminal residues recognized by this antibody. In contrast, the anti-EGFP antibody recognizes the unfused EGFP as well as all the three PRC1 fusion proteins. (D) The distribution of the PRC1-EGFP fusion HAMNO protein in HeLa cells. During interphase, the overexpressed PRC1CEGFP protein (green) induces the formation of circular filaments around nuclei (DNA, red). In common with endogenous PRC1, overexpressed PRC1 is also in nuclei. During mitosis, the PRC1CEGFP protein is associated to a normal spindle (b), and then becomes associated to the midzone (c) and midbody at late telophase (d), similar to endogenous PRC1. The perinuclear ring filaments are evident in interphase (a) and at the end of telophase (d) but are dispersed during mitosis. Bar, 10 m. In accord with previous findings (Jiang et al., 1998), native PRC1 is intranuclear in interphase (Fig. 1 A, a), and then associates with the spindle in early mitosis, being more enriched on the interdigitating MTs, and finally to the spindle midzone in late mitosis (Fig. 1 A, b and c). We have compared the distribution of PRC1 with that of TD-60, a passenger protein that localizes to the spindle midzone in late mitosis (Andreassen et al., 1991). Double immunofluorescence staining with autoimmune antiserum recognizing.
