Mitosis

New Publication in Cell! The Phosphoregulation of Mitosis

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We are incredibly excited to announce that our SnapShot is out today in Cell!
This snapshot of mitosis collates hundreds of phosphorylation events and directly links them with their regulatory kinases and counterbalancing phosphatases, in both time and space, in a highly innovative ‘circtanglar’ cell layout. More importantly, the static PDF version is accompanied by an interactive website that enables users to access direct links to PubMed, UniProt, and Aquaria 3D protein structures for each and every phosphorylation event shown. The pop-up boxes also contain over 100 additional phosphorylation sites on dozens of proteins essential for mitosis. You can access the interactive web version here:  http://www.cell.com/cell/enhanced/odonoghue2
Even better news is that until August 04, 2017 the PDF version of the SnapShot is freely accessible for everyone at the following link https://authors.elsevier.com/a/1VDWh_278yyILK
A big thank-you to Jenny, Sam, Marcos and Sean for helping me put together what I hope will be an amazing resource for anyone interested in how cells divide and phosphorylation in general.
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New Publication: Hedgehog signaling in small cell lung cancer

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Great news we have a new co-author publication in Oncogene!

This work was in collaboration with Prof. Neil Wakins here at the Garvan Institute and focuses on the role of Hedgehog (Hh)  signaling in small cell lung cancer (SCLC). Small cell lung cancer is a common, aggressive malignancy with universally poor prognosis.

Full details can be found here [link]

TITLE: “The role of canonical and non-canonical Hedgehog signaling in tumor progression in a mouse model of small cell lung cancer”

ABSTRACT:

Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.

 

Switching off Cancers Diversity

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JCS paper

A defining feature in over 2/3rds of all solid tumours is the continual loss and gain of whole are small parts of chromosomes. This instability, or CIN for short, strongly implicated in tumour initiation, progression, chemoresistance and poor prognosis. CIN is created through failures during mitosis, whereby whole or parts of a chromosome are segregated incorrectly, thereby created daughter cells with unequal chromosome numbers. Consequently, understanding how mitosis is regulated is essential for uncovering the mechanisms allowing CIN to arise and drive cancer. In our recent publication, we discovered the mechanisms controlling the key regulatory pathway critical to ensuring cells exit mitosis correctly. At the centre of this pathway is a gene call MASTL (short for ‘Microtubule Associated Serine/Threonine Kinase-Like’). The primary function of MASTL is to ensure that the cellular breaks (the phosphatase PP2A), is turned off during mitosis so that the accelerator (Cdk1 kinase) can drive the cell into mitosis. Much like a car, having the accelerator and breaks on at the same time is a bad idea, unless you like the smell of burning rubber. To successfully exit mitosis, and to perfectly segregate chromosomes, the cell must take the foot off the accelerator and turn on the breaks. Because MASTL is the central regulator ensuring the breaks are coordinated with the accelerator, it is essential to understand how MASTL is controlled. To this end, we uncovered that MASTL must be rapidly turned off to allow cells to exit mitosis, and this inactivation is carried out by another cellular brake call PP1 phosphatase (Rogers et al, JCS 2016). Now that we have identified and mapped this novel mitotic exit switch, we hope to be able to shed new light on how CIN drives the initiation and evolution cancer. We believe that with further study we will be able to better predict patient response to chemotherapy, and also identify new ways to ‘switch off’ highly unstable tumours, thereby improving treatment for patients that currently have a very poor prognosis.

Image of Interphase HeLa cell stained for Actin (red), DNA (blue) and the co-localisation of MASTL and PP1 by Proximity Ligation Assay (PLA; green).
Credit: Sam Rogers and Cell Division Lab

 

AntiOxidants and Cancer: A complicated story

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A recent high profile publication in Science Translational Medicine proposed that antioxidants might increase the rate of metastasis in mice models of melanoma.

NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells but did not affect their proliferation. Both antioxidants increased the ratio between reduced and oxidized glutathione in melanoma cells and in lymph node metastases, and the increased migration depended on new glutathione synthesis. Furthermore, both NAC and Trolox increased the activation of the small guanosine triphosphatase (GTPase) RHOA, and blocking downstream RHOA signaling abolished antioxidant-induced migration. These results demonstrate that antioxidants and the  system play a previously unappreciated role in malignant melanoma progression.

This goes against the common idea that anti-oxidants are cancer fighters!

So what is going on?

The answer, much like a Facebook relationship status, is “its complicated”. In fact anti-oxidants can have a wide range of effects on cells, including mitosis. Many “dietary antioxidants Resveratrol and Fisetin (found in red wine), inhibit Cdks, induce a G2 arrest and prevent entry into mitosis” (Burgess et al 2014). Furthermore, we recently showed that partial inhibition of Cdk1 can dramatically disrupt to mitosis. This caused increase cancer cell death… but also increased the rate of chromosome mis-segergations. (McCloy et al 2014). These mitotic errors can drive chromosome instability (CIN), which inturn can lead to the evolution of more aggressive, invasive tumours. Understanding the genetic background of each individual cancer will be key to determining why some cancer cells die and others thrive when given antioxidants.

If you would like to know more on how common stresses such as oxidation can disrupt mitosis, you can read our recent review Stressing Mitosis to Death.

Until then, if you have cancer and are thinking of taking antioxidants, make sure you consult your oncologist as they can significantly affect the efficacy of some chemotherapeutics, and hence maybe doing more harm than good.

 

 

 

New Review Article Published!! “Mechanisms Regulating Phosphatase Specificity During Mitotic Exit”

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icl31035-toc-0001

Great News, we have a new review article that has just been published online today in Inside the Cell!
Its Open Access, so that means its free for everyone to read!

During mitotic exit, phosphatases reverse thousands of phosphorylation events in a specific temporal order to ensure that cell division occurs correctly. This review explores how the physicochemical properties of the phosphosite and surrounding amino acids affect interactions with phosphatase/s and help determine the dephosphorylation of individual phosphorylation sites during mitotic exit.

The Full Reference and link for the Article can be found below:
Samuel Rogers, Rachael McCloy, D Neil Watkins and Andrew Burgess Mechanisms regulating phosphatase specificity and the removal of individual phosphorylation sites during mitotic exit Inside the Cell [Link]

New Paper Published! More data on the global phosphorylation changes during early mitotic exit

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Figure 1

Great news, we have another publication. This time its some extra data left over from our large mass spectrometry study we published in August in Molecular & Cellular Proteomics.

This latest work we provide additional analysis of our large proteomics dataset and identify motifs that correlated strongly with phosphorylation status for each of the major mitotic kinases.

These motifs could be used to predict the stability of phosphorylated residues in proteins of interest, and help infer potential functional roles for uncharacterized phosphorylations.

If you would like more information you can check out the full paper here [Link]. And the great news is that its OpenAccess and FREE for everyone!

Rogers, S., McCloy, R. A., Parker, B. L., Chaudhuri, R., Gayevskiy, V., Hoffman, N. J., Watkins, D. N., Daly, R. J., James, D. E., and Burgess, A. (2015) Dataset from the global phosphoproteomic mapping of early mitotic exit in human cells. Data in Brief 5, 45–52

 

 

 

Using ImageJ to Measure Cell Fluorescence

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Image J can be downloaded for free from here .
This guide can also be downloaded as a complete PDF here: Measuring Cell Fluorescence using ImageJ

Here is a very simple guide for determining the level of  fluorescence in a given region (e.g nucleus)

  1. Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
  2. From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
  3. Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
  4. Now go and select a region next to your cell that has no fluroence, this will be your background.
    NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
  5. Repeat this step for the other cells in the field of view that you want to measure.
  6. Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
  7. Use this formula to calculate the corrected total cell fluorescence (CTCF).
    NB: You can use excel to perform this calculation for you.
    CTCF = Integrated Density – (Area of selected cell  X Mean fluorescence of background readings)

     
  8. Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.

How to Cite this if you wold like to:

We have used this method in these papers:

McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]

Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107: 12564–12569

But you can also find a similar method published here:

Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543

And here:

Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206

And my apologies to any others that I have not mentioned.

Our Latest Publication Accepted and Now Online!

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Great news our latest publication “Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs” has been accepted by the top Proteomics Journal Molecular & Cellular Proteomics.

You can currently download the unformatted version for free here [link]

And here is an still image from the paper showing live HeLa cells undergoing forced phosphatase dependent mitotic exit. The red colour is Histone H2B tagged with the fluorescent mCherry protein, and the Green is tubulin tagged with GFP (green fluorescent protein).

 

HeLa cells undergoing phosphatase dependent mitotic exit
HeLa cells undergoing phosphatase dependent mitotic exit

 

Public Talk “Killing Cancer One Cell at a Time ” now on YouTube

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Here is a recent talk I gave to some members of the public at the Garvan Institute of Medical Research.

It is a very general and simple over-view of explaining 1) how cells in your body proliferate, 2) how this goes wrong in cancer, 3) the challenges we are facing in treating and killing cancer, and 4) most importantly how we hoping to improve current treatments in the near future.

A big thanks to all the fantastic Garvan Foundation Team who hosted, filmed, and edited the event.