JCB

JCB/NYAS Meeting: Chromosomes

Rebecca Heald (UC Berkeley), one of JCB’s senior editors, kick-started Thursday afternoon’s session on Chromosomes.   Heald’s lab is broadly interested in understanding different aspects of cell division from chromosome organization to spindle assembly.  Most of her work is done in an in vitro system using crude extracts purified from eggs of the South African clawed frog Xenopus laevis.  Remarkably, these extracts can support most aspects of the cell cycle in a test tube.  This frog won’t win any beauty contests but watching spindle assembly in these egg extracts is a beautiful sight!   The system provides some unparalleled advantages, namely it is open to biochemical manipulation and different cell cycle stages can be studied independently.  In her talk, Heald described two ongoing projects in her lab on chromosome architecture and organelle size. 

Chromosome condensation is a hallmark of mitosis and crucial for accurate segregation.  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.

Next, 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 21^st issue of JCB.

Tarun Kapoor (Rockefeller University) spoke next about his lab’s interdisciplinary efforts to study properties of the spindle.  Kapoor has done some important work relating to the cell biology of disease.  As a postdoc, he characterized a small molecule inhibitor of the kinesin Eg5 and evaluated its effects on spindle assembly (published in the JCB).  Kapoor started off his talk by mentioning that biotech companies are developing the use of kinesin inhibitors as chemotherapeutics.  He then moved on to discuss his more recent work on spindle architecture.

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.

The fourth talk of the session was from 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.

The last talk of the session was from Wim Vermeulen (Erasmus Medical Center).  He focused on a different aspect of genome maintenance: DNA repair, particularly nucleotide excision repair (NER).  There are many human disorders, such as the UV hypersensitive disorder Xeroderma pigmentosum, that are associated with mutations in genes involved in NER.  NER requires transcription to fill in the excised base and a key factor of the machinery is the transcription factor TFIIH.  Although NER is well understood in vitro, how is it organized in a complex nuclear environment?

Vermeulen described his group’s work on the in vivo dynamics of NER proteins monitored by timelapse imaging and fluorescence recovery after photobleaching (FRAP).  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.

JCB/NYAS Meeting: Stem Cells

Thursday morning's session of the JCB/NYAS meeting focused on stem cells, and began with a talk from long-time JCB editor and recent National Medal of Science awardee Elaine Fuchs, who presented a summary of her lab’s work from recent years. If you’re unfamiliar with the Fuchs lab’s pioneering work on epidermal stem cells (Disclaimer: I’m an ex-Fuchs lab member!), there are a couple of great reviews around to bring you up to speed. Some of the more recent work discussed in this session included the identification of the embryonic stem cell population that gives rise to all the lineages of the skin epithelium (hair follicles, sebaceous glands, and epidermal cells). These stem cells are specified early in embryonic development in a manner dependent on the transcription factor Sox9, and eventually give rise to the previously-identified adult skin stem cell population (Nowak et al, 2008).

Wnt signaling plays a key role in epidermal stem cell differentiation, promoting, for example, stem cell activation or hair follicle formation, depending upon the levels of Wnt activity. Wnt signaling leads to stabilization and nuclear translocation of the transcription factor β-catenin, which acts in conjunction with members of the Lef/Tcf family of transcriptional activators and repressors. The Fuchs lab recently studied Tcf3 and Tcf4, which are co-expressed in epidermal progenitor cells and surprisingly found that these two transcription factors also have Wnt-independent functions in skin homeostasis, helping to maintain stem cells in an undifferentiated state (Nguyen et al, 2009).

Fuchs is, of course, very excited at the prevalent use of adult skin cells as the starting material for induced pluripotent stem (iPS) cell generation, so it was fitting that her presentation was followed by a talk from Alex Meissner. He’s been at the forefront of this burgeoning field as both a student/postdoc with Rudolf Jaenisch and as a group leader at Harvard University and the Broad Institute – you can read more about his background and research interests in our recent profile article. Differentiated adult cells can be reprogrammed into multipotent iPS cells with just a handful of transcription factors. Meissner is interested in understanding the mechanism by which the reprogramming occurs, in order to improve the process’ efficiency. To do this, he’s studied the epigenetic profiles of both fully and partially reprogrammed cells, identifying the DNA and histone modifications corresponding to successful reprogramming.

Meissner is also using similar approaches to determine how similar iPS cells really are to embryonic stem (ES) cells (the answer seems to be “very, but with some small differences”). The most stringent test for pluripotency is a test called tetraploid complementation, which unambiguously demonstrates whether an iPS cell can develop into a live animal. This hurdle has just been cleared for mouse iPS cells (Zhao et al., 2009) but such tests are obviously not possible for human iPS lines. Studies on the epigenomes of human iPS cells will therefore be particularly important for monitoring both the efficiency and efficacy of reprogramming. To this end, Meissner is involved in efforts to produce a reference epigenomic map of human ES cells – this is revealing a lot of variation between different ES cell lines, meaning that there is a lot of work to do before we can draw conclusions about the quality of human iPS cells.

Next came a talk from Fiona Doetsch (Columbia University), who discussed her studies on adult brain stem cells. The presentation largely covered two recently published stories from the Doetsch lab: firstly, the identification of a vascular niche for stem cells in the neural subventricular zone (SVZ). SVZ stem cells are astrocytes that directly contact blood vessels and seem to receive activating signals from the blood due to specializations in the blood brain barrier in this region of the brain (Tavazoie et al., 2008). Secondly, Doetsch described the isolation of micro RNAs that regulate the differentiation of these neural stem cells. One of these, miR-124, is highly expressed in neuroblasts (developing neurons), and overexpressing the RNA promoted neuroblast formation. Knockdown of miR-124, on the other hand, kept SVZ cells dividing, but reduced their differentiation into neurons. miR-124 regulates the timing of SVZ cell differentiation by promoting cell cycle exit and inhibiting stem cell self-renewal. It does this by downregulating a number of targets identified by the Doetsch lab, including the transcription factor Sox9 (see Cheng et al, 2009 and cf. Elaine Fuchs’ epidermal progenitor cells above!).

Irina Conboy from The Berkeley Stem Cell Center was next up to the podium. Conboy is fascinated with aging – she began her talk by comparing the lifespans of rats (3 years) and squirrels (40 years). This huge difference between two ostensibly similar animals suggests to Conboy that aging is not simply controlled by the accumulation of damage over time. Conboy works on muscle, which repairs itself easily when young but not so well as time goes by, even though the number of stem cells (aka satellite cells) remains the same. Conboy has shown that the balance between Notch and TGFβ signaling is critical to determining whether the satellite cells can activate and expand to repair tissue damage (Carlson et al., 2008) and it now seems that a similar mechanism is at play in human cells too. Old muscle can be rejuvenated by restoring the Notch/TGFβ balance, either by knocking down SMADs (the downstream effectors of TGFβ) or by elevating Notch expression.

Parabiosis experiments (in which the circulatory systems of young and old mice are connected together) have shown that systemic factors in the old animal’s blood can prematurely age young muscle stem cells. TGFβ is elevated in the sera of older mice and humans, but Conboy showed that this signaling molecule isn’t acting systemically to inhibit regeneration. TGFβ only seems to act locally in determining aging – the hunt for the systemic factor that ages the stem cell niche goes on (Carlson et al., 2009). You can find out more about Irina Conboy’s work in an upcoming profile article, which will appear in the October 5th issue of the JCB.

The final speaker in the session was Michael Buszczak, from UT Southwestern. Buszczak is interested in histone modifications that silence genes in Drosophila germline stem cells (GSCs), and has used a high throughput “protein trapping” approach to identify proteins involved in GSC maintenance and differentiation. (GFP is randomly inserted throughout the genome, and fly embryos are screened for GFP-tagged proteins with interesting localization and expression patterns). One gene that Buszczak identified in this way is a histone deubiquitinating enzyme called Scrawny (Buszczak et al., 2009). Scrawny silences genes in GSCs (maintaining them in an undifferentiated state) by removing ubiquitin from histone H2B, which would otherwise serve to recruit RNA Pol II. Intriguingly, the human homolog of Scrawny is overexpressed in human ovarian cancers.

Buszczak is now complementing this random search for chromatin modifiers by systematically knocking out enzymes such as histone demethylases and deacetylases. Targeted knockouts in Drosophila are still fairly laborious, even using the new recombineering approach developed by Hugo Bellen’s lab so, in the meantime, Buszczak has begun analyzing a few existing mutants for GSC defects. Buszczak described one histone demethylase mutant that has a non-cell autonomous function in GSC differentiation.

The five talks covered a lot of ground but the connections to the rest of the meeting were clear. Stem cells, and the transcription factors and epigenetics that regulate them, are increasingly relevant to understanding disease from aging to cancer.

JCB Meeting: The Cell Biology of Disease

Our meeting at the New York Academy of Sciences is now underway. There's been some great talks so far - look out for blog updates with more details.
In the meantime, here's a snippet of JCB editor-in-chief Tom Misteli's opening remarks from yesterday evening:

Last chance to register for the JCB conference!

The Journal of Cell Biology Meeting, Cell Biology of Disease: Chromosomes, Cancer, and Stem Cells starts tomorrow (Sept 23-25th). You can still register online, or if you're local, just turn up and register when you get there.

We have two great keynote sessions to start and conclude the meeting, and three focused plenary sessions on Chromosomes, Cancer, and Stem Cells. Our speaker line-up is fantastic, but you don't have to take my word for it, take a look at our program and all the meeting details here.

There will be a reception, and poster session, and also a career panel with speakers from a Bioimaging facility, a tech transfer office, a scientific program officer from the NYAS, and an Editor and a Science writer from the JCB.

We partnered with the New York Academy of Sciences to bring you this event. The NYAS has a spectacular location on the 40th floor of 7 World Trade Center in downtown Manhattan, with incredible views all around the city.

New issue: September 21st

There's a new issue of the JCB published today. Our cover features just one of the many striking images in Nowak et al.'s paper on cell shape and packing. The cells in question are lens fiber cells - fascinating epithelial cells that elongate over a thousand-fold during development to wrap around the lens in concentric layers, appearing as incredibly regular, tightly-packed hexagons in cross-section. Nowak et al. find that this packing is disrupted in mice lacking tropomodulin1, a component of the spectrin-actin network that underlies and supports the fiber cell membranes. I really recommend taking a look at this paper (if only to see the beautiful immunofluorescence and electron microscopy images!), but you can also find out more in my summary of the paper, or by listening to the latest biobytes podcast, also out today.

Elsewhere in this issue, we have two sets of companion papers. The first pair, by Edwards et al. and Sumakovic et al. both describe how the small GTPase Rab2 controls the maturation of dense core vesicles in worm motor neurons. Worms expressing mutant versions of Rab2 are virtually paralyzed because critical component(s) of the vesicles are lost before they can be secreted across the synapse. It remains to be seen precisely what gets lost from Rab2-mutant vesicles, but you can find out what each group thinks might be going on by reading our In Focus article on the subject here.

Our second pair of companion pieces are from Lo et al. and Kemmler et al. Both of these articles reveal the step by step assembly of the 60S ribosome in yeast: a phosphatase called Yvh1 displaces a subunit of the pre-ribosome complex in the nucleus and is then itself displaced by a different subunit as the translation-competent 60S ribosome forms in the cytoplasm.

Meanwhile, Li et al. describe the steps that lead from ER stress to cell death. While short-term ER stress induces the unfolded protein response to allow cells to recover, prolonged stress leads to apoptosis, a process involving expression of the transcription factor CHOP and calcium release from ER stores. Li et al. show how these events are connected: CHOP drives expression of an ER-resident oxidase called ERO1- that, in turn, activates the IP3R calcium channel and subsequent calcium release. As senior author Ira Tabas points out, ER-stress and CHOP-induced apoptosis plays a role in several diseases, including atherosclerosis and diabetes, so understanding how the process occurs is an important step forward.

Returning to yeast, DeVay et al. investigate how a protein related to the endocytic GTPase dynamin promotes mitochondrial fusion. Mgm1 comes in two isoforms: a long, membrane tethered version that is proteolytically cleaved to yield a short, soluble form. Both forms are required for the fusion of mitochondrial inner membranes (a different dynamin-related protein controls outer membrane fusion). DeVay et al. find that both the long and short versions of Mgm1 are targeted to the inner membrane by the mitochondrial-specific phospholipid cardiolipin. Moreover, as summarized here, cardiolipin stimulated the oligomerization and GTPase activity of short-Mgm1, driving membrane fusion. On the other hand, the GTPase activity of long-Mgm1 is dispensable for membrane fusion. Instead, it probably acts as a tether, linking mitochondrial membranes together before short-Mgm1 catalyzes their fusion.

All of this has to be balanced by mitochondrial fission to maintain proper organelle size and distribution. Another dynamin-related GTPase called Drp1 promotes the division of both mitochondria and peroxisomes. As Wakabayashi et al. show, the mitochondria of mice lacking Drp1 are hugely elongated compared to the short, fragmented appearance of wild type mitochondria:

Image from Wakabayashi et al., 2009

These mice die at embryonic day 11.5, while brain-specific Drp1 knockout mice have cerebellar defects, demonstrating the in vivo importance of organelle fission.

Lots of other interesting articles in this issue too, including a review from Anne Simonsen and Sharon Tooze on the crosstalk between autophagosomes and other membrane compartments. You can find the full table of contents by clicking here.

The JCB Congratulates Elaine Fuchs

Elaine Fuchs to Receive National Medal of Science

Today, President Obama honored JCB Editor, Elaine Fuchs by naming her, along with eight others, as recipient of the National Medal of Science. The award will be presented in a ceremony on Oct 7 at the White House.

We congratulate Elaine and her co-recipients for their outstanding contributions and this high honor. For more information see the official press release

New issue and biosights: September 7th

It's a holiday here in the US, but there's still a new issue of the JCB out today. So let's take a look at some of the highlights...

Our cover image this week is taken from the paper by Slattum et al., and shows an apoptotic cell being pushed out of an epithelial sheet. Dying cells can be extruded from either the upper or lower surface of a cell layer; Slattum et al. investigate how the direction of extrusion is determined. As Mitch explains in his summary, a band of actin and myosin assembles in cells surrounding the apoptotic cell and contracts to squeeze the cell out. In order to push a cell upwards, neighboring cells point microtubules to its sides and base, delivering an activator of Rho GTPase that, in turn, activates myosin to put the squeeze on.

Meanwhile, Vale et al. reveal a similar mechanism at play in determining where a cell divides during cytokinesis. They use live TIRF microscopy to show that microtubules deliver the motor protein kinesin 6 (which carries Rho GTPase activators) to the cell equator. This seems to trigger the assembly of myosin filaments which drive cell constriction and cytokinesis. The image below cells the accumulation of kinesin 6 (green) on the plus ends of microtubules (red) at the cell equator:

 

Image from Vale et al., 2009

Also in this issue, Garcia-Cortes and McCollum study the timing of cytokinesis. A fission yeast protein called Etd1 initiates cytokinesis only after the mitotic spindle has become fully elongated during anaphase, ensuring that chromosomes are completely segregated. And Etd1 is destroyed after the cell has divided, in order to switch off further rounds of cytokinesis. You can read more about both of these papers - explaining how cells determine the where and when of cytokinesis - in this month's In Focus.

On a completely different note, Stagg et al. report how cytomegalovirus evades the immune system by triggering the destruction of the MHC class I complex. Two viral proteins fool the infected cell into ubiquitinating the MHC I complex, causing its export from the ER back into the cytoplasm, where it is subsequently degraded by the proteasome. As Mitch describes in his summary, Stagg et al. screened through 373 ubiquitin ligases by RNAi to find the cellular protein responsible for targeting MHC I, namely, an E3 ligase called TRC8.

Also in this issue is a review by Charles Bond and Archa Fox on paraspeckles: ribonucleoprotein particles that regulate gene expression by retaining RNAs in the nucleus. Covering everything from their composition to cellular function, this review tells you all you need to know about this new class of nuclear bodies.

Finally for today, we have a brand new edition of our biosights series of video podcasts. Here, we explore a recent paper from Jung et al., who find that a myosin called myoJ drives the spreading of contractile vacuoles around the actin-rich cortex of dictyostelia, helping the cells discharge excess rain water and avoid osmotic stress. As well as some great videos of this fascinating organelle, senior author John Hammer III explains why the study is an important example of a myosin motor acting as a point to point membrane motor. If you can't see the movie below, you can also watch it by clicking here.