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Comparative Study
. 1998 Dec 28;143(7):1763-74.
doi: 10.1083/jcb.143.7.1763.

INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1

A M Ainsztein  1 , S E Kandels-Lewis, A M Mackay, W C Earnshaw
Affiliations
Comparative Study

INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1

A M Ainsztein et al. J Cell Biol. .

Abstract

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

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Figures

Figure 5
Figure 5
INCENP residues 52–62 constitute a spindle midzone-targeting motif. (A) INCENP1-405:GFP transfers to the central spindle and to midbodies (also see Fig. 2). Subsequent exonuclease III digests produced INCENP1-308:GFP (B), INCENP1-226: GFP (C), and INCENP1-68:GFP (D). Each of these proteins ultimately targeted the spindle midzone. (F) Randomization of the order of amino acid residues 52–62 in INCENP1-405 (52-62r):GFP specifically abolished this transfer. (E) Randomization of amino acid residues 32–44 in INCENP1-405 (32-44r) also abolished transfer to the midzone as a secondary consequence of the fact that this protein did not target centromeres at metaphase (see Fig. 4 F). In all images, the transfected INCENP:GFP fusion protein is shown in green, microtubules are red, and DNA is blue. The arrow in each panel indicates the position of the midbody. The darkened arrows in E and F represent the lack of transfected INCENP at the midbodies. All images are of pig LLCPK cells transfected with chicken INCENP constructs.
Figure 2
Figure 2
INCENP1-405:GFP is released from centromeres back onto the chromosome arms at anaphase onset, but still transfers from chromosomes to the spindle midzone. Transfected LLCPK cells expressing INCENP1-405:GFP (green) were stained for microtubules (red) and DNA (blue). The panels to the right show the INCENP pattern separately in black and white. (A and A′) Prophase. INCENP is distributed along the chromosome arms. (B and B′) Metaphase. INCENP is concentrated in spots at centromeres. (C and C′) Early anaphase. INCENP1-405:GFP is released from centromeres back onto the chromosome arms. (D and D′) Late anaphase. INCENP1-405:GFP gradually transfers from chromosomes to the spindle midzone. By late telophase, all detectable INCENP1-405:GFP has transferred to the midbody (see Fig. 6 A).
Figure 4
Figure 4
The INCENP minimal centromere-targeting module (residues 1–68) contains two highly conserved amino acid sequence motifs required for centromere targeting and spindle transfer. (A) Sequence alignment between the amino-terminal 68-aa residues of chicken, Xenopus, and mouse INCENP. Amino acids that are conserved in all three species are shown in bold type in the line labeled consensus, with similar residues indicated by a black dot. There is 69% sequence identity for residues 32–44 and 100% sequence identity for residues 52–62. (B) Schematic representation of the amino-terminal region of chicken INCENP. The restriction sites used to make deletion and mutant constructs are shown above the bar. The hatched region indicates the centromere-targeting motif. The filled region represents the spindle transfer motif. The boxed sequences below the bar correspond to the wild-type chicken sequences for these motifs. Below these are shown the mutagenized sequences, arrived at by randomization of the amino acid sequences shown in the boxes.
Figure 6
Figure 6
INCENP interacts with HP1Hsα in a yeast two-hybrid assay. Various INCENP deletion constructs were subcloned into the pEG202 vector (Gyuris et al., 1993), and expressed as “bait” in S. cerevisiae. These cells were cotransformed with the pSH18-34 reporter plasmid together with the pJG4-5 vector encoding full-length HP1Hsα. (A) Two clones from each transformation were grown in liquid culture and duplicate samples from each were tested for β-galactosidase activity using ONPG as substrate. Each bait construct is listed at the left of the graph and the relative units of activity are indicated on the x-axis. The numbers to the right of the error bar indicate the calculated activity and the standard error measurements (n = 4). (B) Identification of the region of INCENP that interacts with HP1. Various INCENP constructs in pEG202 were tested for interaction with HP1Hsα using the blue/white overlay assay. +, positive interaction between the INCENP bait and HP1 (specific growth on selective media [Gal-U-H-W-L] and activation of the β-galactosidase gene). −, lack of interaction between the INCENP bait and HP1 (failure of cells to grow on selective media).
Figure 1
Figure 1
INCENP constructs used in this study. The numbers correspond to amino acids and the relevant behavior of the proteins during mitosis is summarized at the right. The thick and thin lines indicate protein sequences that were expressed or deleted, respectively. The balls at the right indicate carboxyl-terminal fusions with A. victoria GFP. The shaded boxes (bottom left) represent the chromo domain (aa 1–68) or full-length HP1Hsα which was fused in frame to INCENP45-839. In INCENP1-405 (32-46r):GFP and INCENP1-405 (52-62r):GFP the amino acid sequences between the arrows were randomized. These sequences are shown in Fig. 5. Due to cloning constraints, INCENP amino-terminal deletion constructs in the pEG202 vector used for two-hybrid analysis (Fig. 7) begin expression of the protein at aa 45 rather than 43. For simplicity, these constructs have been omitted here.
Figure 7
Figure 7
HP1Hsα interacts specifically with INCENP in vitro. Either GST (odd-numbered lanes) or an HP1Hsα:GST fusion protein (even-numbered lanes) expressed in E. coli was bound to glutathione agarose and incubated with in vitro translation reactions containing various INCENP derivatives. The lanes show as follows: INCENP1-839 (lanes 1 and 2); INCENP43-405 (lanes 3 and 4); INCENP43-839 (lanes 5 and 6); INCENP43-405 (lanes 7 and 8); GFP control (lanes 9 and 10); INCENP1-405:GFP (lanes 11 and 12); and INCENP1-68:GFP (lanes 13 and 14). Both panels show autoradiographs.
Figure 3
Figure 3
Amino acid residues 32–44 are required for centromere targeting by INCENP. HeLa cells expressing various INCENP constructs fused to GFP (green) were arrested in mitosis with vinblastine, stained for centromeres with an autoimmune serum (red) and for DNA with DAPI (gray), and visualized by three-dimensional deconvolution microscopy (Agard et al., 1989). Transfected INCENP1-405:GFP, INCENP1-308:GFP, INCENP1-226: GFP, INCENP1-68:GFP, and INCENP1-405 (52-62r):GFP all concentrate at the inner centromere. However, INCENP1-405 (32-44r):GFP fails to localize at centromeres, but instead paints the entire chromosome. In INCENP1-405 (52-62r):GFP and INCENP1-405 (32-44r):GFP, the amino acid sequence indicated was randomized using a mutagenic oligonucleotide.
Figure 8
Figure 8
INCENP lacking the HP1-binding domain moves normally in mitosis. (A and B) Either GST (lanes 2 and 5) or GST: HP1Hsα (lanes 3 and 6) immobilized on glutathione agarose was incubated with in vitro translated INCENP1-839 (lanes 1–3) or INCENP1-839 (Δ69-404) (lanes 4–6). Lanes 1 and 4 show the input of translated protein used in this experiment. A shows the Coomassie blue–stained gel, while B is the autoradiograph. (C–F) Transfected LLCPK cells were stained for INCENP1-839 (Δ69-404) (red), tubulin (green), and DNA (blue). The pattern of INCENP movements shown here is identical to that seen with the full-length INCENP.
Figure 9
Figure 9
Fusion of INCENP to the chromo domain of HP1Hsα bypasses the requirement for INCENP to target to centromeres before transfer to the spindle midzone. (A) Immunoblot showing the protein expression levels of transfected INCENP1-839 (lane B), INCENP43-839 (lane C), and HP1Hsα 1-68:INCENP45-839 (lane D). Equivalent cell numbers were loaded in each lane. Cells transfected with vector alone (lane v) were used as a control. The upper band seen in all lanes is a cross-reactive protein that acts as a loading control. (B′ and B′′) Full-length INCENP1-839 targeted to centromeres at metaphase (B′) and to the midbody in telophase (B′′). (C′ and C′′) INCENP43-839 does not target to centromeres at metaphase (C′) and remains on the chromosomes throughout mitosis (C′′). (D′ and D′′) A chimeric molecule in which the chromo domain of HP1Hsα (aa 1–68) was fused to INCENP45-839 does not target to centromeres at metaphase (D′), but does target to the midbody in telophase (D′′). Identical results were obtained with a chimera containing the full-length HP1Hsα fused to INCENP45-839 (not shown). The figure shows transfected HeLa cells stained for INCENP (red), microtubules (green), and DNA (blue). The arrows indicate the position of the midbody in the telophase cells.
Figure 10
Figure 10
INCENP interacts with the hinge region of HP1Hsα. (A) Schematic diagram of the organization of HP1 together with the various GST-HP1 constructs used in this experiment. In each case, the number at the right represents the gel lanes in which the particular construct was used. The various GST:HP1 constructs were expressed in E. coli, isolated on glutathione agarose, incubated with in vitro translation reactions containing 35S-labeled full-length INCENP1-839, and subjected to SDS-PAGE. This gel was first stained with Coomassie blue (B) and subsequently exposed to film (C). The gel lanes are as follows: i, total in vitro translation reaction; C, GST alone; 1, HP1Hsα 1-190; 2, HP1Hsα 1-68; 3, HP1Hsα 1-110; 4, HP1Hsα 64-111; 5, HP1Hsα 64-190; and 6, HP1Hsα 110-190.
Figure 11
Figure 11
Functional map of INCENP from this and previous studies. The Cen module (residues 1–68) is the minimum region giving centromere targeting. Within this, the 13-aa motif required for centromere targeting (motif C) and the 11-aa motif required for spindle transfer (motif S) are shown. The HP1-binding domain includes residues 135–270, the minimum region of INCENP shown to interact with HP1. The chromosome-binding and microtubule-binding regions were identified in previous studies (Mackay et al., 1993; Mackay et al., 1998).
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