What is wrong with how and when we are taught to write grants

What is wrong with how and when we are taught to write grants

Doing science for a living is one of the most fantastic jobs you can think about. And in general we scientist have a pretty good deal, being at the forefront of new discoveries and getting paid for learning new things. However, for all the great things that go along with it, job security is not one of them. Most of us have faced the situation of being in a great place, working on a  fantastic project, dreaming of making a home in this place, when the reality hits, our contracts run out and without any new grant on the way they have to let you go.

Is it then not strange, that for a sector so dependent on grant money coming in, that scientist are hardly thought how to write grants. As students, all up to the end of our PhDs and even when being postdocs we learn how to do science. From the most basic of how to make up solutions, maintain cell lines, and germinating seeds. To designing and executing complex experiments aimed at answering our questions. We get thought how to write down our results, how to report them to others. Both in writing and in presenting those results in meetings and conferences. During our PhD and while doing our postdocs we get opportunities to supervise others, learning how to lead. We get chances to review papers and to scrutinise the work of other scientists. But the one thing, the most crucial thing on which science relies, applying and with luck receiving a grant to do the research that we are dreaming of, is not thought in any reliable way.

Yes if you are lucky, you might have had an assignment of writing a grant when you were doing your bachelors or master degree. But, who will remember that when they are in the middle of a second postdoc trying to figure out how to get into that magical kingdom in which PIs seem to live.  When you are part of a well financed lab, you would have no need to apply for any kind of grant up to the point that you decide that you have that great idea and want to start your own lab. To be honest, this is not great timing, being thought how to write and apply for a grant or a fellowship at the moment your career depends most on it. It is the one and foremost thing postdocs ask for, teach us how to write and apply for grants. Grant writing courses for postdocs are filled up in no time. Because we know our careers depend on them.

I would suggest a change of practice. Not only should PIs encourage PhDs and Postdocs to apply for travel grants. They also should start a policy in which all new postdocs, will need in their first year, write and apply for a grant or fellowship. In this way postdocs get the training in writing and applying for grants without any real losses when they don’t get them, but with big wins when they do. For those who win the grant it might buy them an extra year or two on their contract. Enough time to cash in on their hard work in the form of some nice papers and preliminary data which they can use in their application for their next grant.

If at the same time PIs introduce a second standard policy, by asking postdocs, for example during those annual review meetings, what they want to do next. And then, of course, not file this information away, but actively help to work out multiple ways for the postdoc to get there, irrespective if this in academia or outside. It might be a way for postdocs to land that fabled staff scientist position they have heard about but never seen advertised. It might be how a postdoc find themselves on an institutes magazine on their way to become and actual science journalist. It might be how as a postdoc you take your results outside the lab and into your start up. And, who knows, it might even be the way how a postdoc finds it way into the magical kingdom of the PIs.

I know this sounds like a lot of extra work for PIs. But, once the first year of postdocs have been initiated in the art of grant writing they are able to help the next who arrives. The same with knowing the passion of your lab members, once known they can be applied when needed. Benefitting both the lab and the person doing it.

Leaf shape development

Leaf shape development

On of the things that intrigues me most in biology is the development of organisms. How does that single cell that is just fertilised knows what to do. To get its polarity established, initiate cell division at the right time, place and direction. What makes it go on developing into recognisable plant and not just a mass of cells. It simply fascinates me. I guess that is what is exciting of the research coming from Enrico Coen lab. I was first introduced to what his lab was doing while working at the John Innes Centre, through our departmental seminars and the annual research days. Here I was introduced to the concepts of how you could compute the development of a leaf.

This they did through painstakingly following the developments of Arabidopsis leaves, tracking cell divisions, from the tiny leaf primordia up to the fully grown leaf. Studying the relationship between cell division, cell size and growth rate. This information they then used to feed into computational models. Which as explained in a recent publication that leaf shape could be brought back to a few parameters and growth factors. The important parameters included growth rate (perpendicular and proximodistal) division rate (division competence and mean threshold cells size). And the growth factors could be brought back to a graded proximodistal factor (PGRAD), a mediolateral factor (MID), a factor distinguishing lamina from petiole (LAM), timing factor (LATE),  and proximal mobile factor (PMF). Interestingly by slightly tweaking the influence these growth factors have an effect on the leaf growth the size of the leaf. This is corresponding to what is observed in mutants with a leaf size phenotype. Another exciting part of this research is that it shows that it is possible to obtain variation in leaf size with just a defined set of growth factors, corresponding to transcription factors in the plant.

These initial studies focused mainly on simple leaves, flat and round. But now they taken this to a whole new level, going from a 2D to a 3D leaf shape. There they show how carnivorous plant carnivorous plant Utricularia gibba adjust their planar (simple) leaves into needle-like structures and cup-shaped traps. First using the same through approach  they used 3D imaging to track cell divisions and to obtain growth rate measurements in three dimensions to use to build a model of how the cup-shaped traps develop. This resulted in a model similar to that of an Arabidopsis leaf, with parameters for growth rate and division rate and growth factors representing a mediolateral factor (MID), ventral midline factor (VEN), and a stalk diameter factor (STK). Using the observed parameters they showed that model was able to reproduce the observed development of an Utricularia gibba cup-shape trap. In addition they showed that by adjusting the parameters they could generate trap morphologies similar to other Utricularia species.

However, a limiting point of the model is in that it considers only the later stages of development of the trap. Not explaining how the curvature of the trap primordium originates. One way of how the initial curvature for the cup-shaped trap might be initiated from a two dimensional origin is through abaxial and adaxial patterning. Comparing the cup-shaped traps with simple leaves it was deducted that the outer side of the cup corresponds to the abaxial (lower) side, and the inner side to the adaxial (upper) side of a simple leaf. Therefor the latest work of the Coen lab investigates the influence of the effect of adaxial and abaxial domains on the polarity field that orientates growth. The decision if cells are part of the adaxial or abaxial site of the leaf occur via gene activity in the leaf primordia. However, solely based on leaf morphology you would not able to distinguish between future leaves or future traps as they are all dome-shaped.

To understand how adaxial and abaxial domain specification might influence trap development the adaxial-abaxial domains in developing traps were identified. While in primordia of leaves the adaxial and abaxial domains each take up approximately halve of the dome-shaped primordia, in primordia of traps the adaxial domain was much smaller than the abaxial domain. To investigate the influence of this on trap development, they build a model in which they could specify the adaxial and abaxial regions. Finding that having an equal adaxial-abaxial distribution would result in either simple leaves or needle like leaves, depending of the settings of the parameters for the different growth rates. However, when they restricted the adaxial region to only a small region on one side of the primordia they found that the resulting leaf would develop into a cup-shaped trap. Furthermore, by adjusting the size and form of the adaxial region, traps of different shapes could be generated.

With developing these models the Coen lab has illustrated the universal mechanism that is behind leaf development. The possibility to generate completely different leaves by adjusting only a few parameters points towards a small group of transcription factors whose differences in expression and activity between species might explain the variation in leaf shapes we see.

Finally, even if you did not understand much about what I have been talking about in this post, I would like to point out that they made lots of movies, both of the models as well as of the imaging they did in order to obtain all the data to feed into the models. You can have a look in the supplemental data of the discussed papers, as well as on the Coen lab website.

Giving a talk

Giving a talk

When preparing to give a talk about your work you always need to make lots of decisions. One is about the amount of background vs results. Ideally you would like to have lots of time to discuss your new results, but for the audience to place them into context, or to understand them in the first place they will need some background. So they will know what you will be taking about. And here lies the difficulty. When you are talking about a widely known topic you might be able to spend a minute or two recapping what everybody already knows, before delving into your results and why they are so exciting. However, when you are studying a highly specialised field, where few people know the ins and outs, it might be wise to spent some more time bringing the audience up to speed. Yes this eats into valuable minutes that you might otherwise spend on talking excitingly about your latest research. But all this excitement will be for nothing if nobody else is understanding what you do. Of course, when you are in a highly specialised field and having an audience from that field you can bring you introduction back to a minute or two.

Giving a talk to a mixed audience with people that know the field well, e.g. your supervisor, and others that are doing completely different things, is tricky. Add to the fact that apparently not only am I speaking about one specialised field but two, phosphoinositides and a biochemistry approach, makes it that I some how lose the audience. Most of the time I forget that taking a biochemistry approach is less common than I think, having always done projects that uses biochemistry in one form or another. So I tend to focus on the new stuff for me, phosphoinositides and what they do. I will be giving these my larger share of background time. I don’t forget to talk through the methods that I used. But, I might forget to expand on why an approach is taken, assuming that it is obvious.

One of the things I apparently should have clarified a bit more in my latest talk, is on why comparing an enrichment in peripheral membrane proteins after salt stress treatment of only 30 minutes would tell us something about the proteins that interact with phosphoinositides under salt stress. The confusion was stemming from the fact that 30 minutes is not long enough for proteins that are synthesised in response to salt stress to be present. It simply takes longer to go through gene activation, transcription and protein synthesis.

PMP and cell
location of peripheral membrane proteins

So it follows that the proteins interacting with phosphoinositides in response to 30 minutes of salt stress are already in the cytosol. In my talk I focussed on that the cytosol contains lots of proteins of which some will be able to interact with phosphoinositides. A lot of these interactions might be aspecific, so an enrichment for specific interactions would make sense. And that the proteins that interact with phosphoinositides are loosely attached to the membrane in the form of peripheral membrane proteins. Therefore, that it makes sense to use a protein extract enriched for peripheral membrane proteins in the interaction assay for identification of the phosphoinositide interactors. What I forgot to clarify was that the peripheral membrane proteins vary depending on the specifics of the cell. So will a non-stressed cell have different peripheral membrane proteins than a stressed cell. Salt stress and heat stress will attract different proteins to the membrane. Just as a cell in the root will have a different subset of peripheral membrane proteins than a cell in a leaf. Knowing this last bit of information and it makes complete sense to have a 30 minute salt stress treatment after which you compare your stressed with non-stressed samples. Not realising this and you might think that after 30 minutes of stress treatment you are still dealing with the same group of proteins as input for your interaction assay.

Now I just have to find a way to wave this into my next talk without it eating up to many precious minutes as I will have lots of exciting results to talk about as well.

SNAREs deal with K+ channels

SNAREs deal with K+ channels

It has been a busy week so it took some time to get my thoughts organised and write this review. Keeping up with vesicle fusion, we now have a look at how SNARE assembly is regulated.

The final step of vesicle fusion with the plasma membrane is regulated by the SNARE complex. This complex is involved in vesicle fusion as follow. Two of the SNARE proteins Qa-SNARE and Qbc-SNARE assemble together with a regulatory SM-protein on the plasma membrane. While the third SNARE protein R-SNARE is located on the vesicle membrane. When the vesicle comes near the plasma membrane the R-SNARE protein is able to interact with the Q-SNARE proteins, bringing the vesicle close to the membrane, enabling fusion. The interaction between Qa-SNARE and Qbc-SNARE is regulated via the open or closed confirmation of Qa-SNARE and its interaction with the SM-protein. However, what causes Qa-SNARE to switch from an closed to open conformation is unclear.

This is were K+ channels come in. Previous research showed that the Arabidopsis Qa-SNARE, SYP121, showed that it binds directly with K+ channels on the plasma membrane, its binding is promoting opening of the channel and K+ uptake. In addition, Arabidopsis R-SNARE, VAMP721, was also shown to bind to K+ channel but instead of promoting K+ uptake it inhibits it. To make matters even more confusing the SPY121 channel binding site overlaps with its SM-protein, SEC11, binding site, which in turn is believed to be important for switching SYP121 from closed to open confirmation.

Now new research from Blatt’s lab is shining new light on the SNARE complex assembly and how its interaction with K+ channels enables coordinating vesicle traffic with K+ uptake. They painstakingly analysed the effects of interaction of  SEC11 and each of the SNARE proteins with the K+ channel. This uncovered that all four proteins are able to interact with the K+ channel, and affect the K+ uptake. With SYP121 and VAMP721 strongly competing for K+ channel binding. However, the presence of SEC11 promotes SYP121 binding with the K+ channel, while inhibiting VAMP721 and K+ channel binding. The interaction of the K+ channel with SYP121 brings about a conformational change that turns SYP121 into an open conformation. Which enabled subsequent binding with SNAP33. They subsequently showed that SEC11 stabilises the binding between SYP121 and SNAP33. And that when SNAP33 is present VAMP721 is able to bind the Q-SNARE complex. The assembly of the SNARE complex finishes with the fusion of the vesicle with the membrane.

The authors summarised their findings and proposed mechanism of SNARE assembly with a nice figure, which I would not like to withhold you, as it probably does a better job showing what is going on than my description.

F10.large
K+ channels KAT1 and KC1 facilitate a binding exchange with SEC11 to promote SNARE assembly for vesicle fusion. The SM protein SEC11 (A) holds SYP121 in the closed conformation . SEC11 interacts with the K+ channels and, with membrane hyperpolarization (+, −), undergoes a three-way binding exchange between SEC11 and SYP121. The Qbc-SNARE SNAP33 (B) stabilizes the complex of SYP121, SEC11, and the K+ channel to moderate the open channel, Qa-SNARE, and SM protein conformations (Fig. 9). Recruiting the R-SNARE VAMP721 (C) facilitates final assembly of the SNARE core complex and transfer of channel binding (D) to SNAP33  while relaxing channel gating and conductance (Fig. 9). Finally, disengaging channel binding with the cis SNARE complex (E) is followed by SNARE complex disassembly. Red arrows by each step in the cycle indicate the nominal channel activity for K+ uptake and its anticipated enhancement with the open conformation of SYP121 prior to assembly with VAMP721.  Reproduced from Waghemare et al., (2019).
Literature

Waghmare et al., K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic. Plant Physiol. Nov 2019, 181 (3) 1096-1113