Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells.

The inner centromere protein (INCENP), which has previously been described in chicken, frog and mouse, is required for correct chromosome segregation and cytokinesis. We have identified the human INCENP gene by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized it to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. Recently we showed that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Here we demonstrate that, consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and we show here that HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines.

Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the arp1 minifilament pointed end.

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

Dynactin is required for microtubule anchoring at centrosomes.

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein-dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein-dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome-lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, gamma tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin-cargo interactions.

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.

Molecular analysis of the INCENPs (inner centromere proteins): separate domains are required for association with microtubules during interphase and with the central spindle during anaphase.

It has recently been proposed that mitotic chromosomes transport certain cytoskeletal proteins to the metaphase plate so that these proteins are able to subsequently participate in the assembly of the anaphase spindle and the cleavage furrow. To understand how such proteins accomplish their dual chromosomal: cytoskeletal role, we have begun a molecular and functional analysis of the inner centromere proteins (INCENPs), founder members of the class of "chromosome passenger proteins". cDNA clones encoding the open reading frames of the two chicken INCENPs were recovered. The predicted proteins, class I INCENP (96,357 D) and class II INCENP (100,931 D) are novel, and differ from each other by the inclusion of a 38-codon insert within the class II coding region. Transient expression of the chicken INCENPs in mammalian cells confirms that the signals and structures required for the transfer of these proteins from chromosomes to cytoskeleton are evolutionarily conserved. Furthermore, these studies reveal that INCENP association with the cytoskeleton is complex. The amino-terminal 42-amino acid residues are required for transfer of the INCENPs from the chromosomes to the mitotic spindle at anaphase, but not for binding of INCENPs to cytoplasmic microtubules. In contrast, an internal 200 amino acid coiled-coil domain was required for association with microtubules, but dispensable for spindle association. These experiments suggest that proteins required for assembly of specialized cytoskeletal structures during mitosis from anaphase onwards might be sequestered in the nucleus throughout interphase to keep them from disrupting the interphase cytoskeleton, and to ensure their correct positioning during mitosis.

Nucleotide sequence of the mouse alpha 1-acid glycoprotein gene 1.

In a previous paper we presented evidence for the existence of at least two alpha 1-acid glycoprotein (AGP) genes in the mouse. One of the cDNA clones characterized in those studies was used to isolate several unique AGP genomic clones. In these studies we present the complete sequence of one of the mouse AGP genes. The sequence analysis includes 595 base pairs (bp) 5' to the site of initiation of transcription and 135 bp 3' to the polyadenylation signal. This mouse AGP gene, designated AGP-1, has six exons, a structure similar to those of the AGP genes in rats and humans. Analysis of the sequence has revealed a number of potential regulatory sites. These include a run of alternating purine-pyrimidine bases [(GT)N] at +2890 to +2945, flanked by three potential glucocorticoid receptor binding sites within intron 5. Two of these TGTTCT at +3069 to +3074 and +3082 to +3087 flank the (GT)n track at its 3' end, and one, which is oriented in the opposite direction (AGAACA), at +2771 to +2776 flanks the track at its 5' end. A longer version of the glucocorticoid receptor site, GGGTACAATGTGTCCT, has been located in the 5' flanking region of the gene (-94 to -79); the sequence AGAACA is another potential glucocorticoid receptor site oriented in the opposite direction and located at -127 to -122. This entire region, from -146 to -42, in the mouse has a strong homology (approximately 85%) to the 5' flanking region of the rat AGP gene, which contains a 78-bp fragment (-120 to -42) that represents the minimal sequence required for glucocorticoid regulation.(ABSTRACT TRUNCATED AT 250 WORDS)