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8th Garvan Signalling Symposium – Registration Now Open !!!

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GarvanSignalling20168th Garvan Signalling Symposium

 Date: Monday 31st of October and Tuesday 1st of November 2016
Venue: The Garvan Institute of Medical Research, Sydney
Registration – Abstract submission to 15th of September 2016
 The Garvan International Signalling Symposium is a premier meeting focused on the mechanisms of signal transduction. It began as a small meeting organised by Prof. Roger Daly around the visit of Prof. Axel Ullrich to the Garvan Institute in 2001. Since then, ‘Signalling Meetings’ have been held every 2-3 years and the meeting has grown into one of the Garvan Institute’s premiere scientific events. This boutique symposium highlights cutting-edge developments from the Asia-Pacific region and the rest of the world.
The Garvan International Signalling Symposium is a premier meeting focused on the mechanisms of signal transduction. It began as a small meeting organised by Prof. Roger Daly around the visit of Prof. Axel Ullrich to the Garvan Institute in 2001. Since then, ‘Signalling Meetings’ have been held every 2-3 years and the meeting has grown into one of the Garvan Institute’s premiere scientific events. This boutique symposium highlights cutting-edge developments from the Asia-Pacific region and the rest of the world.

We welcome scientists at all levels, including students, post-docs, research staff and senior lab heads. The intimate nature of the meeting and enjoyable social functions promotes a collegial atmosphere and excellent networking opportunities. A poster session will be held on the Monday afternoon with generous prizes. Slots have been reserved for short (15 minutes) talks to be selected from submitted abstracts.

The meeting is held at the Garvan Institute in the glamorous Darlinghurst region of Sydney, close to the city, Oxford Street, King’s Cross and the harbour.

This years exciting program features state-of-the-art technologies to investigate a wide range of diseases including cancer, immunology, neuroscience and metabolic disorders.  Special sessions focus on in vivo/intravital signalling, proteomics, control of gene regulation and the structural basis of signalling.

Click Here for more information and to register

 

Speakers

Keynote Speakers

Klaus M Hahn

Klaus HahnThe University of North Carolina at Chapel Hill, USA

Dr. Hahn’s laboratory develops new means to visualize and control protein activity in vivo, and uses them to study the role of signaling dynamics in immune cell decision making. His laboratory has produced broadly applicable approaches to fluorescent biosensors that report conformational changes of endogenous proteins, fluorescent dyes to visualize protein activity in vivo, and protein analogs that can be controlled by light or small molecules. Current biological studies focus on phagocytosis, platelet production and metastasis.

Dr. Hahn studied at the University of Pennsylvania and the University of Virginia, where he received his Ph.D in Organic Chemistry. He was a postdoc at the Center for Fluorescence Research at Carnegie Mellon University, became an Associate Professor of Cell Biology at the Scripps Research Institute, and then moved to UNC-Chapel Hill, where he is the Thurman Distinguished Professor of Pharmacology and Director of the UNC-Olympus Imaging Center. Dr. Hahn is a recipient of an NIH Transformative Grant, the NIH’s James Shannon Director’s Award, and is a fellow of the AAAS. His lab’s work on biosensors was named one of the “10 Breakthroughs of the Decade” by Nature Reviews Molecular Cell Biology.

http://www.hahnlab.com/

 

 

Owen J Sansom

Owen J SansomThe Beatson Institute, Scotland

Owen Sansom is interim director of the Cancer Research UK Beatson Institute, Glasgow. Owen gained his PhD in 2001 working on in vivo models of apoptosis in cancer. Since then, he has been instrumental in determining the molecular hallmarks of colorectal cancer (CRC), including showing the roles of the tumour suppressor protein APC and the WNT signalling pathway and the involvement of intestinal stem cells in tumourigenesis. In 2005, Owen established his own laboratory at the Cancer Research UK Beatson Institute, where he became Deputy Director in 2010. The Sansom laboratory uses in vivo models and 3D in vitro models to recapitulate CRC and pancreatic cancer to investigate the molecular mechanisms underpinning tumourigenesis and to identify novel drug targets. In 2007 Owen won the BACR/AstraZeneca Young Scientist Frank Rose Award and in 2012 was elected a Fellow of the Royal Society of Edinburgh and was awarded the Cancer Research UK Future Leaders in Cancer Research prize.

http://www.beatson.gla.ac.uk/invasion-and-metastasis/owen-sansom-colorectal-cancer-and-wnt-signalling.html

 

International Speakers

Eric O'Neill

Eric O’Neill

University of Oxford, UK

Eric O’Neill is a Senior Group Leader and Associate Professor at the CRUK/MRC Oxford Institute for Radiation Oncology. After completing a Ph.D. at the University of Umeå, Sweden he was a post-doc at the University of Oxford. Subsequently, he was awarded a Marie Curie research fellowship and completed a 5-year post-doctoral position investigating oncogenic and tumour suppressor signalling at the CRUK Beatson Institute for Cancer Research in Glasgow. He is a member of the Association for Radiation Research and an examiner for the Royal College of Radiologists. He has also been on the organising committee for several international conferences.

http://www.radiationoncology.ox.ac.uk/research/eric-oneill

 

PR Shepherd

Peter R Shepherd

University of Auckland, New Zealand

Peter Shepherd gained a PhD in Chemistry from Massey University in New Zealand before post doctoral fellowships at Harvard University and  Cambridge University.  Following faculty positions at University College London, he moved back to Auckland University in 2004 where he is currently deputy director at the Maurice Wilkins Centre, a Centre of Research Excellence. His work focusses on understanding how defects in cell signalling pathways contribute to the development of type-2 diabetes and cancer, particularly focussing on the PI 3-kinase and beta-catenin pathways.  His lab also has a strong translational research focus in the form of several ongoing in house drug discovery projects.

https://unidirectory.auckland.ac.nz/profile/peter-shepherd

 
 
 
 

Tilman Brummer

brummer.png

University of Freiburg, Germany
 

Dr. Tilman Brummer is currently an independent group leader at the Institute of Molecular Medicine and Cell Research (IMMZ) at the Albert-Ludwigs University (ALU) in Freiburg, Germany. He finished his studies in Biology with a diploma at the ALU followed by a PhD thesis on B lymphocyte signalling with Prof. Michael Reth at the Max-Planck-Institute for Immunobiology and Epigenetics. In 2003, he joined the laboratory of Prof. Roger J. Daly at the Garvan Institute of Medical Research in Sydney as a postdoctoral fellow. In 2008, he returned to the newly established Centre of Biological Systems Analysis (ZBSA) at the ALU to establish his independent laboratory funded by the Emmy-Noether program of the German Research Foundation (DFG) before moving to the IMMZ in 2012. He is a principal investigator within the Collaborative Research Centre 850 “Control of Cell Motility in Morphogenesis, Cancer Invasion and Metastasis”, the “Spemann Graduate School of Biology and Medicine” and the “Centre for Biological Signalling Studies” BIOSS. Recently, he has been awarded a prestigious Heisenberg fellowship of the DFG.

The Brummer laboratory is interested in the organisation of intracellular signalling pathways and how their intricate control becomes disturbed in human cancer. The laboratory is particularly interested in understanding the regulation of the BRAF and GAB2 oncoproteins and how they contribute to metastasis and drug resistance in solid tumours and leukaemia.

 

 

Jen Morton

Jennifer Morton

The Beatson Institute, Scotland
 

Jen Morton is a joint leader of the pancreatic cancer research team at the Cancer Research UK Beatson Institute.  Her research focuses mainly on: (a) Investigating the importance of mutations found in human pancreatic tumours using mouse models, (b) Profiling different genetic subsets of pancreatic cancer to better understand the disease and identify specific targets for therapy, and (c) performing preclinical trials of targeted therapies in clinically and genetically relevant pancreatic cancer mouse models.

 

Pat Caswell

Pat Caswell

Wellcome Trust Centre for Cell-Matrix Research, UK

Patrick is based within the Wellcome Trust Centre for Cell Matrix Research at the University of Manchester. Patrick studied Biochemistry at the University of Nottingham, before undertaking a PhD at the University of Leicester and a postdoc in Jim Norman’s lab at the Beatson Institute for Cancer Research. Patrick set up his lab in 2010, focussing on how cell-matrix interactions through integrins generate signals that control key cellular processes such as cell migration, differentiation and survival. The lab is specifically interested in vesicular trafficking, and has recently shown that endocytic trafficking of integrins and co-cargo receptors controls the spatial activation of RhoGTPases to modulate the actin cytoskeleton in invasive cancer cells.

http://www.wellcome-matrix.org/research_groups/pat-caswell.html

 

Vinay Tergaonkar

Vinay Tergaonker

Institute of Molecular and Cell Biology, Singapore
 

Vinay Tergaonkar obtained his Ph.D. (2001), from National Center for Biological Sciences, Bangalore. During his graduate studies he was awarded an international cancer society (UICC) fellowship for collaborative research at Tufts University, Boston, USA. He has been a fellow (2001-2004) and a special fellow (2004-present) of the Leukemia and Lymphoma Society of America and conducted his postdoctoral studies at the Salk Institute for Biological Studies, La Jolla, California. He joined the Institute of Molecular and Cell Biology (IMCB) in late 2005 as Principal Investigator and became a Senior Principal Investigator in 2010 and Research Director in 2015. He is also a Professor at School of Medicine at National University of Singapore. He has been invited to speak at various international venues and meetings such as the Barossa and Hunter valley meetings in Australia, Genes and Cancer meeting in UK, The Argentine Pharmacological society meeting in Buenos Aires, Aichi and Japanese Cancer Society meetings in Japan and the Keystone Symposia. He serves on 
Editorial Boards of 1) Molecular and Cellular Biology (American Society for Molecular Biology) 2) Biochemical Journal (Portland Press) 
3) Critical Reviews in Oncology/Hematology (Elsevier Press), 4) BMC Research Notes (Biomed Central) and 5) Telomeres and Telomerase. He has received international recognition for his work including the British council development award (2014) and the 2015 Premiers’ fellowship from Government of South Australia.

 

Daniel Schramek

Daniel Schramek

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Canada

Daniel Schramek is a Principal Investigator at the Lunenfeld-Tanenbaum Research Institute, and Assistant Professor in the Department of Molecular Genetics, Faculty of Medicine, University of Toronto. He obtained his BSc and MSc in Molecular biology from the University of Vienna and undertook his master thesis under the supervision of Prof. Roger Daly at the Garvan Institute in Sydney. He undertook his PhD work under the supervision of Prof. Josef Penninger at IMBA in Vienna, followed by postdoctoral studies with Prof. Elaine Fuchs at the Rockefeller University in New York. In 2015, Dr. Schramek was recruited to the Lunenfeld-Tanenbaum Research Institute, where he holds an endowed ‘Kierans & Janigan’ Cancer Research Chair as well as a tier 2 Canadian Research Chair in Functional Cancer Genomics. Dr. Schramek was awarded a prestigious HFSP Career Development Award, the Early Researcher Award from the Ontario Ministry of Research & Innovation as well as a CIHR Foundation grant.

His lab focuses on functional cancer genomics and has generated various in vivo CRISPR-gene editing methodologies to screen for novel tumor suppressors, cancer vulnerabilities as well as therapy resistance genes in various mouse models of human cancers. Through this program, Dr. Schramek specifically aims to identify molecular and cellular mechanisms that regulate epithelial tissue growth in adult homeostasis, cancer and cancer-associated inflammation.

http://www.lunenfeld.ca/researchers/schramek

http://schramek.lunenfeld.ca

 

 

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Position available: 2016 Honours Student Project in our Lab

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Great news we are currently looking for a new honours student for 2016.

The title of the project is “Developing novel biosensors to monitor DNA damage in cancer cells”.

Its a very exciting new project incorporating cutting edge microscopy and fluorescent biosensors.

If you think you have what it takes and are interested please feel free contact myself, or UNSW SoMS.
For more information on the UNSW honours program please visit: http://medicalsciences.med.unsw.edu.au/students/soms-honours/

Below is an example of the images that will be created during the project.

Using Thresholds to Measure and Quantify Cells in Image J

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I often get asked how to uses Thresholds to measure things in Image J.

There are some great guides on the web explaining how to use Thresholds in Image J, and here are a few that are well worth checking out [Link1][Link2].

Below are some of the Basic Steps for using Thresholds:

  1. Open your image and duplicate it (Image>Duplicate)
  2. On the duplicate go to Image>Adjust>Threshold
  3. Play with the sliders until all of your cells are red.
  4. Click ‘Apply’
  5. You should now have a ‘binary’ black and white image
  6. Now go to menu Process>Binary and select ‘fill holes’
  7. You may also want to select erode, dilate, open or close to optimise the binary image so that you have nice solid filling of your cells.
  8. Now go to menu Analyse>Set Measurements. Select all the things you want to measure.
  9. Critical steps: make sure that you select your original image (not the binary) in the ‘Redirect to:’ pull down Menu
  10. Also make sure the ‘Limit to threshold’ checkbox is ticked and also tick the ‘Add to overlay’ and ‘Display label’.
  11. Click ok to close the ‘Set Measurements’ box.
  12. Now go to Analyse>Analyse Particles
  13. Here you will need to play around with the size and circularity settings (bit of trial and error) in order to get accurate identification of your cells or ROIs. I suggest making duplicates before you start so that you can quickly try different things to see which one works best.
  14. Make sure you have the Display results tick box selected.
  15. Once you click ok you should have a the measurements box appear with all your measurements for each cell.
  16. You can copy and paste these into Excel or what ever program you like to use.
  17. Go get a coffee and cake you deserve it!

Good luck!

 

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

 

Congratulation to Sam our PhD student on his 1st Author Publication!

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Great news, we recently published a collaborative paper in the journal Cell Division with the Lab of Dr Liz Caldon here at the Garvan.
The title of the paper is “Cyclin E2 is the predominant E-cyclin associated with NPAT in breast cancer cells”, and you can find it online here

It also marked the inaugural 1st Author publication for our PhD student Sam!

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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.

Immunofluorescence Guide

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The Mitchison Lab has an excellent guide on staining and fixing cells for Actin and Microtubules which is worth reading [Link]

Coverslips

Most coverslips come with a fine film coating to stop them sticking to each other. This can reduce the ability of coating agents such as poly-L lysine from working properly, and can thus reduce the ability of cells to properly adhear to the glass. As most mitotic cells ’round’ up and have a much weaker attachment, a poorly coated coverslip can dramatically reduce the numbers of cells you finally have to look at down the microscope. Thus it is always important to first clean the coverslips and then coat them with either Histogrip, Fibronectin, or Poly-L-lysine.

Cleaning Coverslips
1) Boil coverslips in dH2O in a large beaker for several minutes in a microwave
2) Add HCl to a final concentration of about 1M to the hot water. Careful of fumes do in a    fume hood if possible.
3) Cover the beaker with some parafilm, and gently stir/rock the coverslips on for 4-16h or until cool.
4) Rinse the coverslips several times in dH2O.
5) Then rinse 3-5x with 100% Ethanol, leave coverslips in EtOH and go to TC hood
6) In TC hood, separate individual coverslips out onto large piece of clean Kimwipe or similar blotting paper and allow to air dry.
7) They can now be stored (for unto a year) in a 10cm plate or 50ml falcon until coating. Some people like to autoclave them but it is not necessary.

Coating Coverslips
Histogrip (Invitrogen)
1) In a TC hood, make a 1/10 dilution of the histogrip into 100% Acetone in a 50ml Falcon tube. Normally 10-15ml final volume is plenty.
NB: most TC plastic plates will be dissolved by the acetone, but most 50ml Falcons should be ok, but check first.
2) Have a second empty 50ml falcon ready.
3) Drop about 10-20 individual coverslips one by one into the 50ml falcon with the Histogrip solution. Re-cap and invert tube gently several times.
4) Decant the Histogrip solution into the empty 50ml falcon.
5) Place coated coverslips into a 3rd Falcon full of TC clean H2O
6) Repeat steps 3-5 until you have coated enough coverslips
7) Remove H2O and wash coated coverslips 3x with H2O
8) Separate individual coverslips out onto large piece of clean Kimwipe or similar blotting paper and allow to air dry.
9) They can now be stored (for unto a year) in a 10cm plate or 50ml falcon.

General Buffers

PHEM Buffer
25 mM HEPES
1 mM EGTA
60 mM PIPES
2 mM MgCl2
pH = 6.9
(Add in this order.)

Antibody Blocking Solution (ABS)
1X PBS
3% BSA (or 5% Fetal Calf Serum)
0.1% Tween-20

Mix well and filter, aliquot and store at -20°C

Formaldehyde Fixation in PHEM buffer
(Good general use fixation, good for kinetochore proteins, ok for microtubules)

1.    Wash coverslips 2x with 1X PBS.
2 .   If staining a cytoplasmic protein or if you have high background then try a short pre-permeabilize of cells using 0.1-0.5% Triton in PHEM buffer 30 sec -1 min at room temperature (RT).
3.    Carefully fix cells with 3.7% Formaldehyde (fresh is best) diluted in PHEM + 0.5%      TritonX-100 for 10 min at RT.
4.    Wash 4x with 1X PBS at RT. Can be stored at 4°C for 2-3 days at this stage.
5.  Block coverslips for 15-30 min in ABS, then proceed to Antibody Staining

Fixation with -20°C Methanol
(Good for microtubules and most proteins)

1.  Wash coverslips 2x with 1xPBS
2.  Remove all PBS (but do not allow the cells to dry), and immediately add enough -20°C methanol to cover the coverslips. About 2-3ml if using a 6 well plate.
3.  Put the plate in a -20°C freezer for 5 min (NB: can be stored for weeks as long as you keep the coverslips covered in MeOH).
4.  Remove coverslips from MeOH, and rehydrate them in 1xPBS with 0.1-0.5% Triton-X-100 for 10-20 min
5.  Block coverslips for 15-30 min in ABS, then proceed to Antibody Staining.

Antibody Staining

1.    Incubate with ABS  for 15-30 min at RT.
2.    Incubate with 1°Ab diluted in Cell Blocking solution in moist chamber.
3.    Wash 3x 1X PBS-Tween for 5 min at RT.
4.    Incubate in 2°Ab +DAPI in PBS-T in moist chamber.
5.    Wash 3x 1X PBS-T 5 min at RT.
6  .  Mount with Prolong Gold or Mowiol mounting medium on clean glass slide.

Mowiol 4-88 Mounting Medium

Mowiol 4-88 is a high-quality mounting medium with good anti-fade characteristics. It hardens and matches the refractive index of immersion oil, and thus is particularly suited for this form of microscopy. Additional anti-fade (DABCO) is added to further retard photobleaching.

Mowiol 4-88 (Calbiochem; 475904), DABCO (Sigma; D-2522)

1.   Add 2.4g Mowiol to 6g glycerol and stir briefly with a pipette.
2.   Add 12ml dH2O and stir at room temp for several hours or overnight.
3.   Add 12ml 0.2M Tris (pH 8.5) and heat to 50oC for 1-2 hrs while stirring.
4.   When the Mowiol has dissolved, clarify by centrifugation @ 500 x g for 15mins.
5.   Add DABCO to 2.5% (0.72g), aliquot and store at -20oC. Bubbles can be removed by centrifugation. Aliquots can be stored for up to 2 weeks at 4°C or frozen to -20°C for months

Were the front cover feature image on this months issue of Cell Cycle !

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Some more good news to coincide with today’s official release of our manuscript, one of our images has been chosen to be the feature image on the front cover.

It’s a great honour, one that I am very proud of, and is the first time I have ever had a front cover !
You can view the current issue (Volume 13 – Issue 9 – May 1, 2014) here.

Or jump directly to our paper here

Front Cover 

FrontCoverCC13-9

 

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