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

What makes a binding domain

What makes a binding domain

One of the projects that I am busy with at the moment is writing a review about phosphoinositide binding domains. Preparing for that I have been reading old reviews on the same topic. One thing that I noticed was that some domains which have shown to bind phosphoinositides, like the CRAL-TRIO domains, were not included. The logical explaination for that will be of course that they were identified later than said review, but this was not the case. I decided, therefor, to do a bit of research into what makes a binding domain.

While the definition of a domain is clear such as shown on the InterPro website where the define a domain as follow:

“Domains are distinct functional, structural or sequence units that may exist in a variety of biological contexts.”

What defines a binding domain is harder to find, but Wikipedia tells us:

“A binding domain is a protein domain which binds to a specific atom or molecule, such as calcium or DNA. […] Binding domains are essential for the function of many proteins. They are essential because they help splice, assemble, and translate proteins.”

Under this definition we could classify all domains that have proven to function in the binding of molecule such as a lipid or a protein to be called a lipid- or protein-binding domain. This is clear and simple. So why then are some domains for which specific binding capacity is confirmed not classified as such in the literature. Two things appear to be playing a role here.

The first is that sometimes the function of the domain is less clear-cut than some articles tend to make out it to be. So there might be some ambiguity. For example, the TUBBY domain binds highly specific to bi-phosphorylated phosphoinositides. However, as part of its function, once released from its phospholipid it can translocate to the nucleus using the same domain for binding to DNA promoting transcription. Therefore, it could be classified as both a phospholipid- and a DNA-binding domain.

The second is that the domain binding behaviour is different compared to that of other binding domains binding the same type of molecule. For example, for the CRAL-TRIO domain, it is described that it binds phospholipid not only by its head group but by engulfing the whole phospholipid, head and tail. In this respect, it stands out from other phospholipid binding proteins which, in general bind the phospholipid by its head only.

So, in the end, the decision to name a domain as say a phospholipid-binding domain appears to be partly arbitrary. Depending on the level of evidence in favour and against it, but also on how easily its function fits within that single definition. Do I now know if I should include a description of the CRAL-TRIO domains in my review? No I don’t. Will I include them? I probably will as part of their definition is that they bind small lipophilic molecules. Meaning, that when investigating lipid protein interactions you should keep them in mind as potential interactors. This is what readers of the review will want to know. Therefor I should tell them all that is known about it.

Vesicle-vacuole fusion – A little less of a mystery

Vesicle-vacuole fusion – A little less of a mystery

Recently, Yvon Jaillais attended me in his tweet of the latest paper of the Raikhel/Hicks lab. I have been wanting to write a review of this paper as it shows some nice research. However, reading the article I also quickly came to realise, that in order to avoid losing you, I needed some visualisation of all the different interactions that take place. So it took me a bit longer than I hoped for to prepare this highlight. As the figures illustrating the different interactions are mine, all mistakes in the interpretation are mine as well. The paper elegantly describe a set of experiments they have done to decipher a bit more of the PVC/vacuole fusion process. Making use of a small synthetic molecule Endosidin 17 (ES17) that disrupts the fusion process, they found how VPS35-RAB7 interaction plays a role in this process.

In plant cells vacuoles have big role in keeping the cell working as it does.Vacuoles are not only used as storage compartments for lots of molecules. They also have a role in recycling cellular components like receptors. And can act as compartments were cellular molecules are be broken down. As you can see vacuoles fulfil a diverse set of functions. To avoid of it all ending in chaos trafficking towards the vacuole needs to be tightly regulated. Vesicles are emerging from different parts of the cell, and those that are destined to fuse with the vacuole membrane (tonoplast) are called prevacuolar compartments, PVCs for short. Currently our understanding in how this fusion to happens is by the following steps:

  • The RAB-GTPase RAB5 binds to the PVCs and is subsequently activated (RAB5*).
  • PVCs-RAB5* recruits the CORVET (VPS8, VPS3, VPS16, VPS18, and VPS33) complex, followed by interaction with VAMP727 and SYP22 of the tonoplast SNARE complex to initiate fusion with the tonoplast.

Additionally

  • Recruitment of Mon1-Ccz1 triggers exchange of RAB5 for RAB7 and subsequently activates RAB7 (RAB7*).
  • RAB7*-PVC recruits the retromer (VPS35), VPS29, VPS26) complex and interacts with the HOPS (VSP41, VSP39, VPS33, VPS18 and VPS16) tethering complex which subsequent leads to fusion withe the tonoplast, through interaction of VPS33 with SPY22 from the tonoplast SNARE complex

Whereby the exchange of RAB5 for RAB7 and subsequent recruitment of the retromer and HOPS complexes can be seen as one of those additional safety checks to avoid unwanted fusions.

PVC-vacuole fusion 1
Our current understanding of PVC-vacuole fusion

In the recent publication of Raikhel/Hicks lab they investigated this last step in more detail. Using PIN2-GFP as a way to visualise vesicle trafficking they found that ES17 treatment affected PIN2-GFP transport to the vacuole. Using reporter lines for different steps of vesicle transport, they narrowed ES17 effect down to PVCs fusion with the tonoplast.

Following this, they went on to investigate to which protein ES17 was binding and which interaction it disrupted. This led to the finding that ES17 binds to VPS35 at the location where it normally binds RABG3f (a RAB7 protein). The disruption of the interaction between VPSP35 and RABG3f had as result that VPS35 could not anchor itself to the PVC membrane to assemble the retromer complex. Moreover, VPS35-RABG3f interaction was shown to be a requirement for the recruitment of VPS39 and VPS33 of the HOPS complex to the PVC. Which in itself is needed for the fusion of the PVC with the vacuole.

PVC-vacuole fusion 2
PVC-Vacuole fusion with filled in details of the discussed paper

The authors conclude that the interaction of RAB7 with VPS35 and the retromer complex functions as a kind of checkpoint, after which VSP35 presumably releases RRABG3f, so RABG3f becomes available for the recruitment of the HOPS complex and subsequent fusion with the tonoplast.

Literature

Rodriguez-Furlan et al., Interaction between VPS35 and RABG3f is necessary as a checkpoint to control fusion of late compartments with the vacuole. PNAS, 2019, 116 (42) 21291-21301