INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow.

Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to beta-tubulin via a conserved domain encompassing residues 48-85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.

Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

A dynamic connection between centromeres and ND10 proteins.

ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G(2) phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.

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

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.

A dominant mutant of inner centromere protein (INCENP), a chromosomal protein, disrupts prometaphase congression and cytokinesis.

INCENP is a tightly bound chromosomal protein that transfers to the spindle midzone at the metaphase/anaphase transition. Here, we show that an INCENP truncation mutant (INCENP382-839) associates with microtubules but does not bind to chromosomes, and coats the entire spindle throughout mitosis. Furthermore, an INCENP truncation mutant (INCENP43-839) previously shown not to transfer to the spindle at anaphase (Mackay, A.M., D.M. Eckley, C. Chue, and W.C. Earnshaw. 1993. J. Cell Biol. 123:373-385), is shown here to bind chromosomes, but is unable to target to the centromere. Thus, association with the chromosomes, and specifically with centromeres, appears to be essential for INCENP targeting to the correct spindle subdomain at anaphase. An INCENP truncation mutant (INCENP1-405) that targets to centromeres but lacks the microtubule association region acquires strong dominant-negative characteristics. INCENP1-405 interferes with both prometaphase chromosome alignment and the completion of cytokinesis. INCENP1-405 apparently exerts its effect by displacing the endogenous protein from centromeres. These experiments provide evidence of an unexpected link between this chromosomal protein and cytokinesis, and suggest that one function of INCENP may be to integrate the chromosomal and cytoskeletal events of mitosis.

Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells.

After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.

The centromere: hub of chromosomal activities.

Centromeres are the structures that direct eukaryotic chromosome segregation in mitosis and meiosis. There are two major classes of centromeres. Point centromeres, found in the budding yeasts, are compact loci whose constituent proteins are now beginning to yield to biochemical analysis. Regional centromeres, best described in the fission yeast Schizosaccharomyces pombe, encompass many kilobases of DNA and are packaged into heterochromatin. Their associated proteins are as yet poorly understood. In addition to providing the site for microtubule attachment, centromeres also have an important role in checkpoint regulation during mitosis.

Stimulation of tubulin polymerization by MAP-2. Control by protein kinase C-mediated phosphorylation at specific sites in the microtubule-binding region.

Microtubule-associated protein-2 (MAP-2) is extensively phosphorylated on serine and threonine residues, and such modifications affect various cellular processes, including microtubule dynamics. Although MAP-2 phosphorylation has been studied both in vitro and in vivo, nothing is known about the exact location of phosphorylated sites influencing the strength of MAP-2 binding to microtubules. Because the microtubule-binding region (MTBR) retains virtually all of the binding affinity of intact MAP-2 for microtubules, we focused on understanding the effect of protein kinase C phosphorylation on MTBR binding to taxol-stabilized microtubules. We used bacterially expressed MAP-2 MTBR, containing newly introduced CNBr-cleavable methionyl residues, as well as mass spectrometry and site-specific mutagenesis to locate and confirm assignments of critical phosphorylation sites. We report on the localization and role of phosphoryls on two specific residues (Ser-1703 and Ser-1711) in terms of kinase-mediated control of MTBR-stimulated tubulin polymerization. Ser-1703 is situated in the so-called first inter-repeat, and Ser-1711 is located in the second nonidentical repeat. Upon more extensive protein kinase C action, microtubule binding can be abolished by phosphorylation at Ser-1728, but this effect is conditionally dependent on the phosphorylation state at Ser-1703 and/or Ser-1711. Our results suggest that in vivo binding of MAP-2 to microtubules may be controlled by protein kinase C action at these residues, and the strategy presented in this report may facilitate future studies with other kinases.

Cleavage of bovine brain microtubule-associated protein-2 by human immunodeficiency virus proteinase.

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.