DNA bound to octameric histones can be further compacted by linker histones such as histone H1. In work that was published in the JCB, Heald and colleagues showed that H1 function is necessary for mitosis in Xenopus egg extracts. In its absence, chromosomes become tangled up and do not segregate properly. Heald presented new work comparing the function of embryonic and somatic isoforms of histone H1. They examined whether the different isoforms can substitute for each other and the effects of this substitution on chromosome architecture. Surprisingly, isoforms have different properties and regulation despite extensive homology between them. By analyzing the proteins that interact specifically with one isoform vs. another, they are starting to get a handle on how these linker histones differentially control chromosome structure.
Heald then moved on to talk about her lab’s work on determinants of organelle size, namely the nucleus. Previously in The JCB, Heald and colleagues observed that spindle size was affected by cytoplasmic factors. They showed that spindles formed in egg extracts from the larger Xenopus laevis were bigger than those formed in extracts prepared from the smaller Xenopus tropicalis. Curiously, mixing the extracts from these frogs resulted in spindles of intermediate length. Now they are using this system to understand how the size of the nucleus is specified. Like spindles, mixing extracts from the two frogs results in intermediate sized nuclei. Heald’s lab is trying to uncover the molecular basis of this scaling. Simultaneously, they are investigating another scaling phenomenon, i.e., as development proceeds why do nuclei become smaller? Heald predicts that these two lines of inquiry will converge on the same molecular explanation. Answers to these fundamental questions may prove useful therapeutically, as many cancer cells are characterized by alterations in cell size and nuclear morphology.
Iain Cheeseman (Whitehead Institute) zoomed in on the kinetochore, the protein
platform that connects chromosomes and microtubules during mitosis. The kinetochore has a formidable task: It has to ensure biorientation, i.e. only
allow microtubules from opposite spindle poles to attach to opposing
kinetochores. While trying to achieve
this orientation that is crucial for proper chromosome segregation, the
kinetochore is bombarded by microtubules from all directions. Many incorrect attachments (such as
attachment to microtubules from the same pole) occur, which can have disastrous
consequences (see summary on Pellman’s talk below). In molecular terms, how does this structure
translate these subtle differences in microtubule binding to stabilize the
right connection and fix the wrong ones?
This question is the focus of Cheeseman’s current endeavors.
Cheeseman presented his work on the KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which consists of conserved protein complexes that are part of the outer kinetochore. Cheeseman has previously shown that this network is essential for binding of microtubules to the kinetochore. At the conference, he talked about how Aurora B regulates this network and presented a model for how this regulation could account for the ability of the kinetochore to molecularly sense correct and incorrect attachments. To learn more about Cheeseman’s passion for the kinetochore, check out Ben’s People and Ideas on him in the September 21st issue of JCB.
Kapoor and his colleagues devised a way to image single molecules of fluorescent tubulin incorporated into spindles formed in Xenopus egg extracts. Using this single-fluorophore speckle imaging, they found that in contrast to the textbook view of long microtubules emanating from the spindle poles, the entire length of the spindle is composed of relatively short microtubules. This certainly changes my view of what a spindle looks like. They propose that the short microtubules may turn over quickly but through crosslinking enable force generation over long distances, which would be important for chromosome segregation.
Kapoor moved on to talk about the mechanical properties of the spindle. In this work, he and his colleagues set up a dual cantilever system to exert force on the spindles in extracts (the same extracts that Heald uses, see above). Essentially, this lets them grab spindles and squeeze them. Through this approach, they found that they could deform spindles but upon release, the spindles recovered their shape and size. Interestingly, they could break the spindle if they squeezed it pole-to-pole but not if they compressed the spindle along its width. Once broken, the shape of the spindle recovers but not the size or the elasticity (it becomes stiffer). These biophysical approaches will surely uncover more interesting properties of this structure that can be used to examine its function.
David Pellman (Dana Farber Cancer Institute). It is commonly believed that polyploidy, aneuploidy and other forms of chromosome instability (CIN) in cancer cells occurs via erroneous cell division. However, Pellman pointed out that we still don’t know what causes CIN in human cancers. There are correlations between CIN and both centrosome amplification and incorrect chromosome attachments to the spindle. Pellman set out to test the popular hypothesis that CIN caused by the presence of multiple centrosomes is due to multipolar cell division. They engineered cells to have multiple centrosomes and then observed their mitoses. Interestingly, they found that most multipolar mitoses are transient; the extra centrosomes cluster together and allow ‘bipolar’ spindle assembly. Moreover, multipolar anaphases were rare and if they did occur, the cells died. So how do the extra centrosomes promote CIN? They found that the presence of the extra centrosomes correlated with an increased frequency in merotelic attachments—when both kinetochores are attached to microtubules emanating from the same centrosome. Pellman predicts these attachments only weakly activate the spindle assembly checkpoint and are therefore hard to correct. These chromosomes lag behind the others during anaphase and seem to be the cause of the aneuploidy (for more details see Ganem et al. (2009)). They also found that the lagging chromosomes get encapsulated in a micronucleus. They are now trying to figure out what happens to these micronuclei, another hallmark of cancer cells, and how they contribute to genome instability.
last talk of the session was from Wim Vermeulen (
They found that in cultured cells, these proteins are not preassembled into a complex but rather are distributed in the nucleus. When damage occurs, they undergo rapid and sequential assembly at the damage site. But what about the situation in vivo? To address this, Vermeulen’s group engineered a mouse expressing YFP-tagged TFIIH. These mice appear normal and Vermeulen presented some of their preliminary results examining NER in vivo in different tissues. This mouse will be a valuable tool to monitor and visualize the molecular dynamics of transcription and repair in the presence of experimental and genetic manipulations that trigger disease.
Overall, this session was a really stimulating discussion of how compromising genome integrity can lead to disease.