combining-mitotic-cell-synchronization-high-resolution-confocal

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Northwestern University

Robust but dynamic attachment between kinetochores and spindle microtubules is an essential prerequisite for accurate chromosome segregation and for preventing aneuploidy. A pair of recent studies has shed light on the details of how the molecular machinery that orchestrates these attachments is recruited to mitotic kinetochores.

Cell Division: Molecular Pathways for KMN Kinetochore Recruitment.

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4(Cdt2), which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4(Cdt2) targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNA(DNA)) and recruits CRL4(Cdt2). Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNA(DNA). Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.

Sequential replication-coupled destruction at G1/S ensures genome stability.

Mitosis entails the bona fide segregation of duplicated chromosomes. This process is accomplished by the attachment of kinetochores on chromosomes to microtubules (MTs) of the mitotic spindle. Once the appropriate attachment is achieved, the spindle assembly checkpoint (SAC) that delays the premature onset of anaphase needs to be silenced for the cell to proceed to anaphase and cytokinesis. Therefore, while it is imperative to preserve the SAC when kinetochores are unattached, it is of paramount importance that SAC components are removed post kinetochore microtubule (kMT) attachment. Precise knowledge of how kMT attachments trigger the removal of SAC components from kinetochores or how the checkpoint proteins feedback in to the attachment machinery remains elusive. This review aims to describe the recent advances that provide an insight into the interplay of molecular events that coordinate and regulate the SAC activity in response to kMT attachment during cell division.

How the SAC gets the axe: Integrating kinetochore microtubule attachments with spindle assembly checkpoint signaling.

The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.

The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells.

A colossal amount of basic research over the past few decades has provided unprecedented insights into the highly complex process of cell division. There is an ever-expanding catalog of proteins that orchestrate, participate and coordinate in the exquisite processes of spindle formation, chromosome dynamics and the formation and regulation of kinetochore microtubule attachments. Use of classical microtubule poisons has still been widely and often successfully used to combat a variety of cancers, but their non-selective interference in other crucial physiologic processes necessitate the identification of novel druggable components specific to the cell cycle/division pathway. Considering cell cycle deregulation, unscheduled proliferation, genomic instability and chromosomal instability as a hallmark of tumor cells, there lies an enormous untapped terrain that needs to be unearthed before a drug can pave its way from bench to bedside. This review attempts to systematically summarize the advances made in this context so far with an emphasis on endocrine-related cancers and the avenues for future progress to target mitotic mechanisms in an effort to combat these dreadful cancers.

Article	Year
Cell Division: Molecular Pathways for KMN Kinetochore Recruitment. Current biology : CB\| PubMed ID: 25898103	2015
Sequential replication-coupled destruction at G1/S ensures genome stability. Genes & development\| PubMed ID: 26272819	2015
How the SAC gets the axe: Integrating kinetochore microtubule attachments with spindle assembly checkpoint signaling. Bioarchitecture\| PubMed ID: 26430805	2014
The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells. Open biology\| PubMed ID: 26581576	2015
Targeting mitotic pathways for endocrine-related cancer therapeutics. Endocrine-related cancer\| PubMed ID: 28615236	2017

Dileep Varma

Biography

JoVE Journal Publications

Other Publications