Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

Eric C Tauchman,Frederick J Boehm,Jennifer G Deluca

doi:10.1038/ncomms10036

Abstract

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.

Highlights

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores
We previously demonstrated that cultured vertebrate cells expressing a mutant Hec[1] that cannot be phosphorylated by Aurora B kinase on its disordered ‘tail’ domain (9A-Hec1) generate hyper-stable kinetochore– microtubule attachments as evidenced by: (i) increased interkinetochore distances, (ii) thicker kinetochore fibres and (iii) an accumulation of syntelic attachments, in which both sister kinetochores of a pair are attached to a single pole[10,26,29,30]
Number of Mad1-positive kinetochores per cell. It is well-established that formation of stable, end-on kinetochore–microtubule attachments quenches the ‘waitanaphase’ signal generated by the spindle assembly checkpoint (SAC)

Summary

Introduction

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. An issue that still remains unresolved, is whether the presence of stable kinetochore microtubules is sufficient to induce changes in the kinetochore that lead to SAC silencing, or if kinetochore tension is required This issue has been difficult to address, since on chromosome bi-orientation and formation of correct kinetochore–microtubule attachments the development of kinetochore tension is a consequence. In a landmark study by the Rieder lab using PtK1 cells, a single remaining unattached kinetochore was laser ablated, which resulted in silencing the SAC and entry into anaphase[18] In this case, tension between the two sister kinetochores (typically monitored by the distance between kinetochores) was surely lost, pointing to stable microtubule attachment as the critical parameter monitored by the SAC. We find that SAC silencing occurs in the absence of large changes in intra-kinetochore distance, suggesting that substantial intrakinetochore stretching is not required for quenching the SAC signal

Methods

Results

Conclusion