You can see a lot by looking – TIRF

Time for a new series. “You can see a lot by looking” basicaly cool images from our research + a little explanation. Microscopy in its various forms has always been one of my favourite things to do, especially when trying out new modalities that open up opportunities to ask new/deeper questions, so I figured we might as well show off some of our pretty images.

#1 in the series is from today; Lee and I used the new Total Internal Reflection Fluorescence (TIRF) microscope at the University of Liverpool’s Centre for cell imaging. This was the first time we have used this microscope and these are just a couple of a set of really nice images, acquired thanks to the expert help of Dave Mason (@dn_mason).


Why is it cool? Well, TIRF is pretty awesome. Basically, whereas normal microscopy involves illuminating the sample directly and then collecting either the transmitted light or the reflected light depending what you are looking for, TIRF involves illuminating the sample at a shallow angle and collecting the light that is internally reflected  (the same principle that fibre optics work by). The practical upshot of this is that when you image at the critical angle, you effectively limit the illumination to near the cell substrate boundary, the bottom ~75nm (1/750,000 of a mm!). You can see the difference this makes in the images below; left = TIRF, right = conventional imaging. Without the TIRF it is much harder to see the fine organisation of the protein at the bottom of the cell.

How are we using it? Well in the magenta and green image, we have imaged live corneal epithelial cells where we have induced expression of the LaNt alpha31 protein with a green fluorescent tag and laminin beta3 with a mCherry tag (shown in magenta) and while the whole cell expresses these proteins, we have limited what we are looking at to just the point where the cell is touching the glass. So here, where we see codistribution of signal i.e white, it’s showing the LaNt and the laminin are close together at the bottom of the cell. The green only signal, e.g. on the left middle, is where there is lant but limited laminin or vice versa for the magenta. In the image below, I have split one of our other images into its component parts (left=LaNt, right=laminin), in this cell there is a much closer match up in the patterns of the two proteins. Proximity alone doesn’t mean interaction but these data add to the other pieces of the story that we are building about how these two proteins influence each other. FYI the scale bar in the image below is 10 micrometers, 1/100th of a mm, you are looking at just a small part of one cell)

TIRFcr
This ‘scope itself pretty amazing, not only can you do TIRF but also Atomic force microscopy at the same time. Looking forward to the next set of experiments…

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Valentina en route to viva!!!

Hello readers!

It’s Valentina here, the first PhD student to have joined the Hamill’s lab. I am writing my first article on a very special day…on my way to Dundee to defend my thesis! It’s been a really cool adventure, full of incredible discoveries. 

When I have started, the only available data on LaNts were related to the skin; after three years and many exciting experiments we now know the effects of LaNt a31 in the cornea…which may seem only a little organ of the body, but how amazing is it to be able to see the world?! We have loads of beautiful videos of live laminin’s deposition from corneal epithelial cells and we have established a LaNt a31 overexpression animal model, the first one ever! 

Working in this lab has been fantastic and hopefully today I will be able to transmit my excitement to the examiners 🙂 If my work could open at least one more door to future projects and ideas, then I’ll be doubly proud. 

Going on, the Hamill lab, laminins and LaNts are waiting to be completely undercovered 🙂  much more to follow on this exciting topic…

Soon to be (hopefully) Dr. Vale 

They grow up so fast – MRes Spring 2017

April time again and the end of another block of MRes Clinical Sciences projects. This time the Hamill lab hosted three students; Kareem Hassanin, Conro Conro Sugden and Tobi Oyewole. Today the boys had their presentations/poster session.

First up; Conro told his story of developing our new minigene construct to investigate LaNt regulation and testing a few mutant versions of it to prove it works. Conor, with Lee Troughton’s help, did some excellent work; generating flo2017-04-13 15.38.50w cytometry,
RT-PCR, western blotting and fluorescence microscopy images that demonstrate that our new system is effective for studying intron retention and alternative polyadenylation and showing that the exon 9 splice site has a big role to play in determining splicing efficiency. His data has gone straight into a grant application and should form the starting point for the next stage of the LaNt project.

Next, Tobi under 20170413_120630the joint supervision of myself and Colin Willoughby and working alongside Fight For Sight student Thanos, worked on determining the optimal delivery mechanism and conditions for delivering small RNA molecules into corneal epithelial cells. This work is critical first step for the the next stage of Thanos’ PhD studies and, although Tobi was frustrated at times, makes a big difference to the lab. Optimisation is the biggest part of all experiments, without these steps we wouldn’t be able to ask the big questions.

Finally, Kareem working alongside Dr Valentina Barrera and using samples kindly provided by Prof Geor20170413_141809ge Bou Gharios, optimised our new rabbit anti-mouse LaNt antibody staining protocol and then did the first staining of embryos and various tissues in the adult. These are really short projects and filled with frustration as just at the very end, Kareem finally had conditions nailed down to get super clean, specific, staining that has opened up a whole range of new options and research questions. One thousand emails bounced back and forward yesterday as he tried to identify all the structures that were and were not stained. Although super cool, really this is just the beginning; knowing where the protein is automatically generates the question of what it is doing in that location as we predict that LaNt function is context specific, so these data too will lay the groundwork for the next grant(s) and next paper.

All in all, a really productive 3 months. Looking forward to the next block…

 

The wall of LaNt research

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Just back from another conference and Lee has added his recent poster to our growing collection. A quick rearrange and we have the wall of LaNt research!

Yes, we have gone too far, taking over a bunch of the poster boards on our floor but it is so cool seeing the work from the various members of my group coming together into a really big story. I recommend working from left to right as you look at them to get a picture from the gene to the protein to the whole tissue.

It starts at the transcript level with Lee’s poster showing LaNt regulation of laminin expression. Also including images of changing protein expression in squamous cell carcinoma from Valentina Barrera and  Conro’s minigene analysis of critical residues involved in splicing regulation.

Next we move on to some of our cell biology work looking at LaNt a31 in the front of the eye where Vale B demonstrates differential distribution accacross the corneal epithelium and Lee and Valentina Iorio show knockdown and overexpression affecting cell migration/spreading

After that its into the molecular biology, with Vale I using fluorescently tagged LaNts and laminins to study their interplay in corneal epithelial cells in culture and also the effects  LaNt overexpression has on junctional complex assembly and matrix organisation.

In Poster 4 Tobi and Vale B’s work brings us back to whole tissues, this time using limbal explants as a 3D model of corneal wound repair and includes our data demonstrating changes in LaNt distribution as wounds heal.

Finally Umar and Vale B show some quite preliminary work of LaNt roles in angiogenesis and the effect of overexpression/knockdown on HUVEC tube formation.

I will now be sending all new students to study all these posters before starting work in the group. Seems like an easy way to get up to speed!

They grow up so fast…. PhD thesis submission edition!

Today the pride levels are extra high. Valentina Iorio submitted her PhD thesis!!!

It’s in


Valentina was the first person to join my lab. I interviewed her whilst still in Chicago and was super impressed with not only her academic ability but also her bubbly personality. I have never looked back. 

It’s been an amazing three and a bit years; loads of impressive data, some incredibly cool live cell imaging videos of LaNts and laminin and even during the tough times, when things weren’t working and when reviewer two was needlesslessy negative, Vale faced it all with a smile. 

The end result is an excellent very readable thesis. She will defend sometime in June but at this time on behalf of myself, Carl Sheridan and George Bou Gharios;

Well done Vale! We’re proud of you