Septin 7 Interacts with Centromere-associated Protein E and Is Required for Its Kinetochore Localization

Abstract

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (2), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore plays an active role in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3). During mitosis, attaching, positioning, and bi-orientating, kinetochores with the spindle microtubules play essential roles in chromosome segregation and genomic stability.CENP-E 3 is a microtubule-based kinesin motor protein located on the outer kinetochore and is involved in a stable microtubule-kinetochore attachment (4). CENP-E participates in the chromosome movements from prometaphase to anaphase (5) by tethering the kinetochores to microtubule "plus" ends and moving toward the equator (3, 6), thereby helping mono-oriented chromosomes align at the metaphase plate before bi-orientation (7). A recent study showed that the mammalian septin network coop...

Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules

“…Yeast Two-hybrid Assay-Yeast two-hybrid assays were performed as described previously (15,16). Briefly, SKAP cDNA was inserted into the BamHI-EcoRI sites of pGBKT7 vector to create a fusion with amino acids 1-147 of the Gal4 DNA-binding domain (BD).…”

Sep(t)arate or not – how some cells take septin-independent routes through cytokinesis

“…Interestingly, septin 2 and 6 have been detected in the microtubule spindle of HeLa and MDCK cells during metaphase (Spiliotis et al, 2005). Knockdown of SEPT2, SEPT6 or SEPT7 leads to the accumulation of pro-metaphase cells and a significant reduction in the amount of the centromere-associated protein E (CENP-E) at the kinetochores, indicating that the septin core complex might have a more general role as a scaffold at the midplane of the mitotic spindle (Spiliotis et al, 2005;Zhu et al, 2008). Therefore, in addition to their key role in cytokinesis, septins also appear to be essential in mitosis.…”

SEPT7 (septin 7)

“…It does belong to the group of septins with the least members (SEPT7 and 13, only) and is found in almost all of the heterofilaments known till date. It is therefore a basic element for filament formation (Nakahira et al, 2010) Expression SEPT7 has cytoplasmic expression in most tissues but is always found associated to actin filaments as well as several regions through-out mitosis (Zhu et al, 2008). There are already cytoplasmic, nuclear, and overall (total) SEPT7 protein expression levels which have also been determined in PTC (Igci et al, 2014).…”

“…While at anaphase it is associated with the central spindle and at the cleavage furrow in anaphase cells. It has been detected at the midbody in telophase and is associated with actin stress fibers (Zhu et al, 2008).…”