Anaphase regulation

Chromosome bridges in anaphase: Genomic integrity relies on the idea that replication is faithfully completed before chromosome segregation, an idea consistent with the replication of the bulk of chromosomes prior to mitotic entry.  However, ultrafine bridges (UFBs) found in normal (i.e., non-cancer cells) mammalian cells are actively replicated during anaphase and are sensitive to replication stress prior to mitosis (Chan et al, Nature Cell Bio, 2009). 

Our recent work has shown that similar structures form in budding yeast and are sensitive to replication stress. Over 50% of normal cell divisions exhibit extended UFBs in anaphase, arguing that these structures are part of normal cell divisions. 

Chromosome bridge surviellance pathway: A number of studies and our own work support the idea that there is a chromosome bridge surviellance pathway. We are interested in how slow to resolve UFBs are detected and how this pathway coordinates spindle dynamics, sister chromatid resolution and cytokinesis, as reviewed in the below diagram for budding yeast. 


Chromosome bridge detection: Evidence from mutants that result in chromosome mis-segregation and chromatin in the spindle midzone demonstrate that the Aurora B pathway is activated to delay abscission during cytokinesis (Mendoza et al, Nature Cell Biol, 2009). The targets of this particular pathway and the mechanism by which Aurora B is activated are not well understood. It should be noted that Aurora B is known to modify both chromatin as well as spindle-associated proteins, thus the implications for altering Aurora B activity are tied to multiple aspects of chromosome and spindle function. We are interested in understanding the mechanisms that control Aurora B activity under conditions of slow to resolve UFBs. 

Spindle regulation: The anaphase spindle (image on left from budding yeast, DKR and KBK) is a complex protein network made up of overlapping, interpolar microtubules, kinetochore microtubules and an array of associated proteins that bundle overlapping microtubules and control microtubule growth, shrinkage, stability, and force generation. The activity of many of the associated proteins have been studied (e.g., kinesin motors) but it is less clear how they work in a dynamic manner to control spindle elongation, stability, and breakdown. The observation that many spindle proteins are post-translationally modified in a cell cycle dependent manner suggests that the network is highly dynamic.  

The Aurora B chromosome passenger complex (CPC) is thought to be major regulator of both chromatin and microtubules. We recently found that CPCs regulate the distribution of kinesin-5 motors on the anaphase spindles in budding yeast (Rozelle et al, JCB 2011). CPC regulation of motors switches their activity to favor outward sliding when evenly distributed on the spindle or to favor "braking" when motors are clustered at the midzone. 

We are currently exploring the connection between CPC regulation of the spindle and the impact of slow to resolve UFBs on spindle dynamics. Our model is that UFBs alter the activity of Aurora B CPCs and thus alter spindle behavior to ensure proper resolution of chromosome bridges. 

Bridge resolution: The final resolution of sister chromatids requires a number of DNA metabolism enzymes, including the Bloom's helicase and topoisomerase type I. Although the precise molecular mechanisms that ensure resolution are not understood, mutations in the Bloom's helicase result in patients with high rates of genomic instabilty and a predisposition to cancer. Using budding yeast as a model system, we are interested in understanding how bridge resolution is linked to chromatin condensation and spindle forces. Our working model is that slow to resolve UFBs arise from late replication intermediates that trigger feedback, which modulates both chromatin compaction and spindle forces to ensure conjoined sisters are resolved. 

Cytokinesis regulation: Work on the "no-cut" pathway supports the idea that slow to resolve UFBs feedback to inhibit abscission, the completion of membrane deposition at the division plane, during cytokinesis (Norden et al, Cell 2006). However, our prelimary results suggest that UFBs that are excerbated by replication stress do not significantly delay abscission. We are currently pursuing a more careful analysis of this prediction. 

In addition, we are also exploring the idea that persistent UFBs may alter cytokinesis in unique ways. Our previous findings indicate that CPCs regulate septin dynamics (Gillis et al, JCB 2005 and Thomas et al, MCB 2007). Septins are filamentous cytoskeletal elements that are associated with polarized cell growth. In budding yeast, they form a ring at the site of bud growth and also serve as a marker of the division plane. At the end of mitosis, the septin ring in the mother cell is disassembled and then re-assembled at the next bud site (red rings in the diagram). Our work showed that the activity of Sli15 (INCENP) is required for the timely disassembly of septin rings at the end of anaphase. In contrast, the Aurora B kinase (Ipl1) is not required. This separation of function argues that there are distinct CPCs that carry out discrete functions in anaphase (Thomas and Kaplan, 2008). It is possible that septin dynamics are linked to resolution of sister chromatids or UFBs. Our working model is that UFBs trigger CPCs to delay septin disassembly and this in turn prevents the final steps of cytokinesis. 



kbkaplan@ucdavis.edu © K.B. Kaplan 2012