Centrosomes acquire dense microtubule nucleation sites in the beginning of mitosis. Failure of this process (called centrosome maturation) impairs the function of mitotic centrosomes to create robust mitotic spindles, resulting in lagging and scattered chromosomes that subsequently cause abnormal nuclear morphology such as bi- tri- or multiple-nuclei with or without small nucleus, as seen routinely in MDS.
We previously reported that the Miki (LOC253012) gene, located in 7q21.3, is frequently deleted in MDS patients, and that low levels of Miki are tightly associated with abnormal mitosis and nuclear morphology (BBRC 2009). Here we demonstrate that Miki plays critical roles in the formation of robust spindles required for the prompt movement of chromosomes in a poly(ADP) ribosylation (PARsylation)-dependent manner (a part of data was published in Mol. Cell 2012).
While Miki was localized in the Golgi apparatus during interphase, it was relocated to centrosomes at the beggining of mitosis. Treatment of cells with Miki-specific siRNA induced ‘pseudometaphase’ condition, in which lagging chromosomes juxtaposed to, or even situated behind, spindle poles. Pseudometaphase was followed by apoptosis or abnormal exit from mitosis that creates cells with abnormal nuclear morphology. This phenotype of Miki-downregulation was caused by the reduced robustness of mitotic spindles. α -tubulin staining of siRNA-treated cells revealed curling and disorganized spindles, with an occasional chaotic centrosome at only one side of the alignment. In addition, Miki-downregulation reduced γ-tubulin signals in mitotic centrosomes and markedly inhibited microtubule nucleation, shown by the impaired accumulation of the EB1 microtubule tip-binding protein at centrosomes.
In immnunoblot analysis of lysate extracted from isolated spindles/centrosomes using Miki antibody, we detected a dense 125 kDa band in addition to the expected 50 kDa band and found that the 125 kDa band represents PARsylated Miki. A recent report indicated that tankyrase-1, a PAR polymerase (PARP), is required for the progression of prometaphase. We found that the downregulation of tankyrase-1 prevents Miki from localizing to mitotic centrosomes. In addition, immunoblot analysis of immunoprecipitation revealed that Miki is a substrate for tankyrase-1. These data suggested that tankyrase-1 PARsylates and translocates Miki from the Golgi apparatus to mitotic centrosomes/spindles during the short period from late G2 to prophase.
We also found that PARsylated Miki promotes CG-NAP, a major component of microtubule nucleation sites, to concentrate in mitotic centrosomes. Interestingly, the CG-NAP gene resides 1.2Mb centromeric to Miki in band 7q21, and approximately 20% of MDS patients lose one allele of both Miki and CG-NAP genes. This indicates that loss of 7q results in low expression of two crucial factors in the tankyrase-1/Miki-dependent system for centrosome maturation, and that this may cause miotic/nuclear abnormalities and chromosome instability characteristic of 7q- MDS.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.