Just submitted our manuscript on a new live cell reporter for total protein content

Measuring the size of human cells is hard, or requires fancy equipment most people don’t have. Our tool makes it much easier. Our preprint is available here on bioRxiv http://disq.us/t/3cykkni

We express a fluorescent protein from a constitutive promoter so that its amount is proportional to cell size, then measure total fluorescence by flow cytometry or widefield microscopy. We show that these measurements correlate well with those from other established methods including flow cytometry, protein-binding dye, nuclear volume, and dry mass. Crucially, we also show that our fluorescence measurements are more robust and less dependent on image segmentation than is the commonly-used measurement of nuclear volume. Moreover, our straightforward technique, requiring only a wide field fluorescence microscope, does not require equipment unavailable to most cell biology laboratories and is compatible with commonly used live cell imaging methods. Finally, use of two colors allows estimation of the concentration (ratio of your favorite protein to the size reporter) of the second fluorescently tagged protein. Thus, our reporter can be used to facilitate concentration measurement using simple wide field instrumentation available in most cell biology labs.

Nice work Daniel!

The cell cycle inhibitor Rb is diluted by cell growth to trigger the G1/S transition

We just posted it on biorxiv here http://disq.us/t/38qjs6b so it is almost like Evgeny’s work is published… hmm… Comments are welcome as we are just kicking off the submission process.

In any case, I think this is an important piece of work. As we said in the conclusion: “Our work demonstrates the importance of measuring protein concentration dynamics. Previous genetic studies showed that Rb and p107 deletion, as well as cyclin D or E overexpression, reduced cell size, and genome-wide screens have identified additional regulators involved in size homeostasis including Largen and p38 MAPK 7–12,40. However, while deletion and overexpression change the concentration of these key regulatory molecules to affect cell size, these experiments do not imply that the concentration of these molecules change in wild type cells as they grow through G1. Our studies of protein concentration dynamics therefore complement the previous genetic studies by identifying the key regulators whose concentrations are in fact changed by cell growth in G1 to drive cell cycle progression, such as Rb and p107.

Conceptually, our work shows how cell size signals can originate in any part of a pathway or network. For a protein to be a ‘size sensor’ all that is required is that the protein’s concentration changes with cell size, and that this concentration change influences cell cycle progression. Here, we identified the size sensors Rb and p107, which are two cell cycle inhibitors in the middle of the mammalian G1/S regulatory pathway. Following similar logic, we previously identified the cell cycle inhibitor Whi5 in budding yeast as a cell size sensor. While Whi5 is functionally similar to Rb, in that both proteins inhibit transcription factors at the core of the G1/S transition, they share no sequence similarity and have a different evolutionary origin 21. That both these transcriptional inhibitors serve as size sensors by being diluted by cell growth demonstrates a deep conservation of the systems level logic at the core of cell cycle control.”

It really is amazing how useful studying yeast has been, even when it has no right to be. Here, we are simply studying the analogous circuit, which uses completely different proteins (see work from the Buchler lab, e.g., Medina et al in eLife). It is just so exciting to see that circuit ideas are useful and conserved, even when protein constituents of those circuits are not. Next, we need to figure out how this works in live growing animals since that may, of course, be different than the tissue culture models we examined here. Again, cell line studies have been informative, even when they really have no right to be… I know what model my money is on, but, of course, we could well be wrong. We have to do the experiments. Exciting times!

We just posted a manuscript on BioRxiv on how cyclin D find the retinoblastoma protein... it actually might be important

https://www.biorxiv.org/content/early/2018/08/21/397448

Despite all the work so for on cell cycle control, we  don't know too much how cyclins, the specificity subunit of cyclin-dependent kinases, find their targets. Surprisingly, this is also true for cyclin D, which launches the human cell cycle, at least in part by phosphorylating the retinoblastoma protein, Rb. We should know how cyclin D recognizes Rb, and now we do... it might actually be important for human health. The cyclin D-Cdk4,6-Rb pathway is just now becoming an important cancer therapeutic pathway with a new series of drugs in clinical trials targeting the ATP binding pocket of Cdk4,6. That these Cdk4,6 inhibitors have significant off-target activites raises the possibility that a new class of drugs targeting cyclin substrate recognition based on our helix docking mechanism could improve cancer therapy (seriously).

On another note, there is something interesting about the cyclin D recognition mechanism. Other cyclins recognize short linear motifs in unstructured regions of the target proteins, while we observe a cyclin recognizing a structural element (an alpha-helix that resides within a previously reported unstructured region) in the C-terminus of Rb. This expands our understanding of structural elements responsible for determining cyclin-dependent kinase targets to drive the many facets of cell division.

Finally, much remains to be done, for example, we still don't know all the mechanisms cyclins E or A use to recognize Rb too... and we should understand that since these are interactions at the very core of the eukaryotic cell cycle.

My students graduate and Devon Chandler-Brown won the Yanofsky prize for best thesis in molecular biology!

Congratulations to Carolyn Kravitz as well, who finished an excellent senior thesis and will be starting her PhD studies at Yale in the fall. I'm sure it will go great.

Andreas Doncic RIP, a few inadequate words below

These words were spoken in memory of Andreas Doncic in the Lokey conference room at Stanford University on April 25th, 2018:

I wanted to convene here in memory of Andreas Doncic who passed away last weekend in a traffic accident in Dallas.  Andreas was such a magnificent and incredible person. He was larger than life. It seemed so impossible that he could die. When Jon came in my office first thing Monday morning looking like death, I thought for sure he found another site on Whi5 that was phosphorylated in G1 and we were set back a few months in completing his work. Even when Jon told me the words, they made no sense to me.  They are still barely comprehensible. Anyway, I know many of us here have been deeply touched by Andreas’s presence in our lives. So, I thought it would be a good idea for us all to get together and reflect a bit. I will start by saying a few inadequate words to express how much Andreas meant to me and then we can hear form whoever else wants to do the same.

I first met Andreas in the Fall of 2008, just a month or two after starting here as an assistant professor. I had no lab space, no lab, and no results.  We hadn’t yet done an experiment. All I had was enthusiasm and ideas. And he believed in me and in our ideas and chose to come to our small lab, despite offers from several larger established labs in the field. We worked together very hard in setting everything up. Even now, almost 10 years later, so many of our protocols and analysis methods bear his mark. Not a day passes when we don’t still use his software. I have never worked so closely with anyone before, and will never work again so closely with anyone again (now that I really don’t know what I’m doing in the lab anymore).  We spent countless hours together, on weekends, in the evenings. I made the strains, he made the movies and did the image analysis. Just over a year later, we submitted our first paper. While it took a little while for the field to understand the importance of his work, they eventually did, and success, and a string of beautiful papers followed.

Andreas accomplished a lot in his too short career and I want to just highlight a bit how important his work was. Both because it was deeply important to him and because it remains deeply important to me. He was able to combine mathematical ideas from dynamic systems to understand the pivotal decision, know as the start of the cell cycle in yeast, where the decision is made as to whether or not the cell should divide. Unlike most math or physics oriented work, Andreas really took on the biology on its own terms, and tried to understand which concepts applied to promote its understanding, and which did not. We never tried to shoehorn in clever math that wasn’t applicable to further biological understanding. He made important discoveries in how precise the decision was (it was very precise), how signals were processed to give the cell a memory of past events, and how signaling pathways were organized spatially to enhance this memory. This was all based on precise image segmentation and quantitative imaging. He continued this line of work at UT Southwestern in the cell biology department where he started a lab and was applying and further developing these concepts to understand how yeast make decisions to enter meiosis. I recently learned that just last Friday that he received favorable reviews from Molecular Cell for his first paper from his new lab, which was a real tour-de-force technically in that he was imaging 6 colors at the same time in live cells… I am currently working with his department head and will work with his students and postdocs to prepare the resubmission of this manuscript.

Andreas faced many obstacles in our society. While his published work was excellent and led to many interviews, he initially had difficulty converting those interviews to job offers.  I think it was about 15 or possibly more interviews over 3 years before the first offer came in. This was because he was a different and special person, and the interviews we conduct for faculty searches are often superficial. At times he made inappropriate jokes, or just unusual jokes, often times in formal situations, like giving a job talk or the dinner afterwards. He was irrepressible. He was always uniquely himself, and he was judged for it. Yet, despite these setbacks and rejections, which took a toll, he persevered, and fought, and worked only harder, and published more and even better papers.

So much potential. I don’t know what to say – Two years into his faculty search, I began calling anyone I knew at all the places he applied to. I believed in him. When I talked to the people at Southwestern when he was interviewing, I said that there was a zero percent chance that Andreas wouldn’t discover important things and to get things done. He had such a relentless drive. The second thing I told them, was that he would be the best department citizen that they ever had. This also turned out to be true as he was volunteering to teach a matlab class to the incoming graduate students, and had taken on teaching a cell cycle course – way above any med school teaching requirements. I knew this would happen because he was the most generous and giving person in our lab. He would spend countless hours training new graduate students and postdocs in the arts of microscopy and image analysis. And he would do it immediately and without reservation or expectation of anything in return. He was a true believer in the science for its own sake and its ability to shed light on the objective truth in nature. I’ve talked to several faculty at Southwestern and it was clear that they appreciated Andreas for who he was, and, in this brief time there, he had an important impact on them. He was loved in Dallas.

And Andreas was succeeding, finally, in everything he had worked so hard for. He lab was taking off, his young and beautiful family was growing. It seems to me just so deeply unfair that it all ended this way. He had so much promise and was taken from us far too early.

The only solace I think we can take is that I know that Andreas lived his life to the fullest. In 40 years he lived more than most did in 80. Every single day he gave it 200% in all possible ways. I think we can all learn from his example. He will be sorely missed.

Bibliography from Andreas’s time at Stanford

A Doncic, M Falleur-Fettig & JM Skotheim, “Distinct interactions select and maintain a specific cell fate” Mol. Cell 43, 528–539 (2011).

A Doncic, U Eser, O Atay & JM Skotheim, “An algorithm to automate yeast segmentation and tracking” PLoS ONE 8 (3), e57970 (2013).

A Doncic & JM Skotheim, “Feed-forward regulation ensures stability and rapid reversibility of a cellular state” Mol. Cell 50, 1-13 (2013).

A Doncic, O Atay, E Valk, A Grande, A Bush, G Vasen, A Colman-Lerner, M Loog & JM Skotheim, “Compartmentalization of a bistable switch enhances memory across a cellular transition” Cell 160, 1182-95 (2015).

O Atay, A Doncic & JM Skotheim, “Switch-like transitions can modularize complex biological networks”, Cell Systems 3 (2), 121-132 doi:10.1016/j.cels.2016.06.010 (2016).

C Schwarz, A Johnson, M Koivomagi, E Zatulovskiy, C Kravitz, A Doncic & JM Skotheim, “A precise Cdk2 threshold determines passage through the restriction point” Molecular Cell, 69 (2), 253-264, e5 (2018).