Category Archives: conferences & courses

Separating cells is hard

I write this entry to accompany my short talk at Woodstock.bio meeting ( #physiologicalirrelevantconference ) .

A few years ago we published a paper in PNAS in which we showed that full-length mRNAs transfer between mammalian cells via a unique type of structure called membrane nanotubes, or tunneling nanotubes (TNTs). This work was started at Rob Singer’s lab, continued at the Gerst lab and in collaboration with Arjun Raj.

I wrote a “behind the scenes” post, detailing how that paper came to be, and some of the problems I had along the way.

I next published a method paper, which also included some new information – in particular that the transferred mRNA is encapsulated in an unknown protein shell. I wrote a “behind the paper” post at the Springer Nature blogs. There, I told about all the problems I had just because a simple change of the cell fixation conditions of my FISH protocol.

The problem is depicted on the right side of my Woodstock.bio slide:

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Very briefly – because the regular FISH protocol leads to TNTs breakage and loss, I decided to increase TNTs stability by adding glutaraldehyde to the fixation buffer. This led to a four-fold increase in TNT preservation. But the transferred mRNA disappeared! It took me a very long time to figure out what’s going on there and partially solve this – at the expense of TNTs’ stability again.  I still have hopes to find a fixative that will preserve the TNTs without affecting the FISH quality.

The left side of the slide depicts our grad student’s greatest achievement – something we’ve been trying to get at over the past six (6!) years. The idea is very simple – co-culture human and mouse cells. After some time, separate then to pure human or mouse cell populations and send for RNA-seq. This should reveal the entire transferome – which human mRNAs are found in the mouse cells and vice versa. As a control, we have a mix of human & mouse cells which were cultured separately, mixed and immediately separated in parallel to the co-culture.

The issue is that we need very high purity. This is because we estimated the amount of transferred mRNA as 1% or less of the endogenous. So if we have 1% donor cell contamination, it will obscure the transferred mRNAs.

For about 2-3 years, I tried to separate the cells with flow cytometry, using various labeling strategies and conditions. But I never managed to get a clear signal of our positive control (MS2-labeled mouse beta-actin mRNA) in co-culture over mix. Then Sandipan Dasgupta joined our lab and instead of FACS sorting, he used affinity purification with magnetic beads to sort the cells. It seemed to be going fairly well. So much so that we also designed an in vivo experiment in mice. We then sent our samples to sequencing only to find out that the sequencing facility had made some mistakes, or there was another problem and all our samples were either contaminated with mouse RNA, or just mixed somehow. That facility closed (we were last in queue ) so there was no way to solve it. But, we also learnt that we probably would not have had enough coverage anyway.

So, Sandi repeated the (in vitro) experiment in order to collect new samples for RNA seq – but we noticed, based on more quality control experiments we did, that the separation was not good enough for us. Although the mouse cells were very pure (99.9%), the human cells always had a small level of mouse cells (98.5% purity of the human cells). Since our expected signal is about 1-2% of the mRNAs being transferred, we could barely see a signal in co-culture compared to mix (1.3-fold).

So, Sandi worked really hard, playing with the conditions until he solved it, and got consistent 99.9% purity of the human cells – just a few months ago. The qRT-PCR result in the slide shows 4-5 fold more human beta-actin mRNA in mouse cells in co-culture compared to mix (we have similar results for the mouse beta-actin mRNA in human cells). The samples were shipped for deep RNA seq (150 million reads per sample) and we are waiting for the results.

We also have more experiments going on – but these stories are for another time.

Maybe we should open a falafel stand” is an actual text from Jeff when we discussed one Saturday evening on Whatsapp about all the problems we encounter in our experiments.

 

 

RNA meeting 2019 – Part 1

The RNA society meeting is big. With over 150 talks and almost 700 posters, there’s a lot of new stuff to learn. This was my first RNA society meeting and I decided from the beginning to take it easy. That is why I did not live tweet like I usually do.

Instead, I thought of writing a summery blog post.

Continue reading

ASCB15 – part 3

(part 1, part 2)

I ended part 2 Monday night. It was an exciting day with many excellent talks, but the best talk (mine, of course!) was due the next day.

Tuesday started with the seminar on engineering cells and tissues. There was the mandatory CRISPR talk as the great new thing in bio-engineering these days. Jennifer Doudna talked about the discovery, then went on to discuss new experiments (using Halo-tag to track Cas9 in live cell nuclei to study movement & binding kinetics) and improved technologies (transfect cells with pre-assembled Cas9-gRNA for quick editing & less off-target cleavage).

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ASCB15 – part 2

I ended Part 1 after the morning session on pushing the boundaries of imaging.

After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.

After this session, I attended my first “discussion table”. Continue reading

ASCB15 part 1

The ASCB meeting brings scientists from all levels to talk about cell biology, which is actually almost anything “biology”. But there’s also a full program dedicated to other matters, like science careers, science publishing, science communications and science policy. This is also a great venue for companies to show their products, and for organizations/institutions to recruit new members. If I remember the numbers correctly, there were over 550 oral presentations and over 2,700 posters. I overheard someone saying there were ~6000 people attending the meeting. I typically go to RNA meetings that are mostly in the lower 100’s of participants. So, to me, that’s a large meeting.

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Continue reading

Announcements

I am aware that it has been a while since I updated new posts on fluorescent microscopy. I blame life. And work.
Anyway, I have several papers piled up, waiting to get blogged.

In other news, I wrote a blog post for Addgene blog. Addgene, for those of you who do not know, is a non-profit organization that helps biologists share plasmids. Briefly, researchers can send Addgene their plasmids (which were featured in their publications) and other researches can order these plasmids from Addgene at a relatively small fee (relative to the amount invested in cloning a plasmid). My post is about how to choose the best suited fluorescent protein, something I wrote about in this blog sporadically. It should get published soon.

On May 22 I’m going to present my work at the annual meeting of the Israeli Society for Cancer Research.

On July 12-13 I’m going to present my work at the Gordon-Merck Research Seminar on Post-Transcriptional Gene Regulation  and the very next day at the Gordon Research conference on the same topic. You can still apply (for poster presentation only) for both of these.

On the publications’s front, a revised version of a paper I co-authored (which continues my work on the decay-transcription story) was recently sent back to the editors. We’re hoping for good news soon.

Am co-authoring review paper on mRNA localization for Nature Reviews Molecular & Cell Biology. Hope to submit within a couple of weeks.

And, the research I’ve been working on for the past two years is finally starting to take shape as a paper. Hope to submit within a month or so.

Also, for the first time, I’m now an official reviewer of a peer-reviewed journal ( RNA journal ).

iBiology Microscopy Course

Optical Nanoscopy Blog

Light microscopy has become one of the most useful tools in the life sciences. Following the traditions of great courses on light microscopy, such as those offered by the Marine Biological Laboratory, EMBO, and the NCBS in Bangalore, this free online comprehensive course begins with the basics of optics, proceeds through transmitted light microscopy, covers the various methods of imaging fluorescent samples, describes how cameras work and image processing, and concludes with some of the latest advances in light microscopy. In addition to lectures, they also provide labs (filmed at a microscope) and short tips, so as to cover pragmatics of how to use microscopes. Assessments are provided for each lecture. Enjoy learning microscopy!

ibiologyMicroscopyCourseLong

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