Context is everything

Plant & zo

The science of plants and more


Context is everything

I have been reading a ton of articles lately, in order to get a better grip on phosphoinositide-binding proteins. Slowly, this made me realise that how we present our findings is important. Of course a well written article is easier to read than one that is not. But the order we present our findings in and the context we place them in also has a big impact. It not only determines the chance of them to be picked up by others, but also how they will place your results in context with what they already know and what they will learn in the future. I will give you an example of something that I have been coming across while reading about phosphoinositides.
In one paper (1) it starts with

“PIPs (phosphoinositides) greatly influence growth, development, and responses to external cues.”

Followed by

“PIPs are also known to regulate themselves numerous cellular processes, including membrane trafficking, ion channel activity, cytoskeleton dynamics, cell polarity, vacuolar morphology and chloroplast division.”

Reading this you would be thinking wow this molecule is involved in lots of processes, how can it do this?
In another paper (2), actually in the same journal and issue, has actually the opposite approach, here they start with

“both groups of molecules (small GTPases and phosphoinositides) coordinate trafficking between different membranes as specific mechanisms are in place to ensure their correct spatiotemporal distribution.”

They then explain the general mechanisms before going on with

“Finally, to illustrate the aforementioned concepts, we use the examples of vacuolar sorting and polarized trafficking, notably in the context of tip growth.”

Reading this you might think, this is an interesting mechanism, where else does it apply to?
As the examples above illustrate, the way you present the data affects how it is thought about. What is becoming clear is that phosphoinositides are part of membrane trafficking machinery and that membrane trafficking affects a lot of developmental processes. By placing the membrane trafficking machinery first and the affected processes second Noack and Jailais are helping the reader to make the connection that all the mentioned processes are affected by a single mechanism that through fine tuning can influence them all.
Although, placing the affected processes first and the how by the membrane trafficking machinery second would not prevent you from making this connection, it is not helping either. You will need to understand how phosphoinositides affect each of these processes in turn before you are able to see that in each process they are more or less doing the same thing. Asking the reader to do much more work before coming to the same conclusion.
While it is understandable in the process of discovery that initially things are presented as if they are not interconnected. However, once the connection is made it is important that this is not lost in subsequent reporting of new findings. So in the case of the role of phosphoinositides we can say something like this “phosphoinositides are involved in membrane trafficking and regulation of membrane proteins. Through this they affect not only plant growth, but also development and responses to external cues.”

References

1. de Campos and Schaaf, The regulation of cell polarity by lipid transfer porteis of the SEC14 family, Current Biology, 2017, 40:158-168
2. Noack and Jaillais, Precision targeting by phosphoinositides: How PIs direct endomembrane trafficking in plants, current Opinion in Plant Biology, 2017, 40:22-23

How plants avoid salt

Plant & zo

The science of plants and more


How plants avoid salt

Plants don’t like high salt levels in their cells. They do therefore everything they can to avoid this. As mentioned in earlier posts plants have a number of strategies for this. However, the best strategy is not taking up to much salt in the first place. Being sensitive to salts, plants can sense differences in salt concentrations. When the roots grow in a patch of soil high in salt their growth is slower than it would have been when growing in soil low in salt. However, when growing in soil low in salt, but coming across a patch of soil high in salt the roots will adjust their direction of growth to avoid the high salt soil. To understand how plants do this you will need to how root growth is regulated.

root auxin flow normal
Reverse fountain auxin flow in a root grown under normal conditions

Simply said, root growth is a product of increasing the number of cells and increase of the length of the cells. Root growth is regulated by distribution of the plant hormone auxin. Auxin is made in the shoot and transported via the phloem to the tip of the root. Here it creates an auxin maximum, which inhibits cell differentiation and elongation, but creates a zone with fast dividing cells. From the tip of the root auxin is actively transported up again creating a reverse fountain effect. With auxin concentration decreasing the further away the cells are from the tip. This enables the plant to control the differentiation and elongation of the cells, the cells just above the zone with fast dividing cells still have a slightly lower level of auxin, resulting in inhibition of division and elongation, but promotion of differentiation. In the cells above that the auxin level is lower again, now enabling elongation, the lengthening of the cells. When the root is simply growing down the reverse fountain of the auxin gradient is symmetric.

root auxin flow salt gradient
Reverse fountain auxin flow in a root growing in a salt gradient

But when a salt gradient is sensed, the auxin gradient becomes asymmetrically, with higher levels of auxin at the side with less salt. This higher auxin level inhibits elongation in the cells away from the salt. While at the same time at the side with more salt the auxin levels are lower. These lower auxin levels promote elongation in the cells near the salt. This asymmetric lengthening of the cells bends the root away from the salt. Enabling the root to grow away from the salt.

root beding salt gradient
Root growing away from high salt levels

Active auxin transport is needed for the creation of the reverse auxin fountain. It was found that transporters called PIN are responsible for auxin transport. Under normal circumstances they are mainly located in the plasma membrane at the top of the cell. When exposed to salt the cell use endocytosis to remove these PIN proteins from the plasma membrane, reducing the auxin flow. So when the exposure to salt is asymmetrically, there will be more PIN proteins in the plasma membranes of cells on the side with less salt than in the plasma membrane of the cells on the side with more salt. Resulting in the asymmetric auxin gradient, and subsequent bending of the root.

References

Galvan-Ampudia et. al., Halotropism is a response of plant roots to avoid a saline environment, Current Biology, 2013, 23: 2044-2050

Abas et al., Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism, Nature Cell Biology, 2006, 8: 249-256

Brunoud et al., A novel sensor to map auxin response and distribution at high spatio-temporal resolution, Nature, 2012, 482: 103-106

Band et al., Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism, PNAS, 2012, 109: 4668-467

How phosphoinositol lipids can have an effect

Plant & zo

The science of plants and more


How phosphoinosotol lipids can have an effect

So in the last post I talked about that I am busy with trying to identify phosphoinositol lipids. But did not really say why I would like to know which proteins bind to my phosphoinositol lipid of interest. Phosphoinositol lipids can been seen as markers of the membranes. For example the plasma membrane has the phosphoinositol lipids PI(4)P and PI(4,5)P2. but membranes of the Golgi has only PI(4)P. In this way the cell is able to distinguish between the Golgi and the plasma membrane. With as result that the proteins whose interaction should happen at the plasma membrane are doing their job at the plasma membrane and not at the Golgi membrane. As such phosphoinositol lipids function as signalling molecules. In cells local differences in the abundance of phospholipids have been seen, also their abundance can be affected by external stimuli, for example salt stress increases PI(4,5)P2 levels. Thereby specifying the location and timing of the interactions that take place. Of which the exocyst complex is one.

The exocyst complex helps connecting secretory vesicles to the plasma membrane. The secretory pathway can basically divided into five stages, starting with the budding of a vesicle, its transport towards the target membrane, initial attachment of the vesicle to the target membrane, the docking of the vesicle and finally fusion with the membrane. The exocyst complex is involved in the third stage, the initial attachment of the vesicle to the plasma membrane. By doing so it also creates a bigger anchorage point where all the proteins that are needed for successful exocytosis.

The exocyst complex was originally discovered in yeast and is and consist out of eight proteins. Homologues of these eight proteins are present in plants, however as for a lot of plant genes, some have multiple paralogues which can complicate things a bit. Two of the exocyst subunits, Sec3 and EXO70, create contact points with the plasma membrane. Sec3 does this by directly binding to PI(4,5)P2. Although it is known that EXO70 binds to the plasma membrane the specific phosphoinositol lipid it binds to is unknown. Furthermore there are multiple paralogues of EXO70, suggesting they play a role in specificity of the location of binding to the plasma membrane. The binding of the exocyst to the plasma membrane is further stabilised by the binding of Sec10 to the cytoskeleton.

For the binding of the vesicle to the exocyst complex it is less clear how this might be happening. It is known that Sec15 plays a role in binding the vesicle to the exocyst complex. However Sec15 is not binding directly to the vesicle lipids like Sec3 and EXO70 are doing at the plasma membrane. Instead, vesicles use RabE GTPases to bind to the exocyst. These GTPases are attached in the membrane of the vesicle via an lipid binding domain. For binding to the exocyst RabE first binds the SCD complex which in turn bind Sec15. At the same time the SCD complex activates RabE, enabling it to activate PIP5K which phosphorylates PI(4)P to produce PI(4,5)P2. As there are multiple paralogous for RabE, SCD as well as for PIP5K, it is possible they have a role in the specificity of the location of vesicle excretion. However not much is known how exocyst subtypes select the vesicles they bind.

Hopefully this made it a little bit clearer how a signalling lipid located in the plasma membrane can actually have an effect. The next question that everybody will ask of course is will those exocyst complex proteins be among those proteins that bind my phophoinositide lipid in response to salt stress. To be able to make a prediction like that we should look about what we know is happening during salt stress to a cell. The initial response will be a loss of water, due to osmosis, shrinking the cell, as well as taking up Na+ and Cl ions. But at the time of sampling the cell is already trying to make up for this by actively pumping water in and pumping salt out. For this ion channels need to be activated, this can possible go via phosphoinositide lipids. It is also possible that these ion channels are newly deposited at the plasma membrane via exocytosis. Then it is a possibility that EXO70 and Sec3 are among the proteins that are identified as interacting with the phosphoinositide lipids during salt stress. But this is pure speculation based on what we guess is going on at the time of analysis. The only way to find out is actually identifying those proteins that bind my phosphoinositol lipid.

Literature

Saeed et al., Dissecting the plant exocyst, Current Opinion in Plant Biology, 2019, 52: 69-76

Noack and Jaillais, Precision targeting by phosphoinositides: How PIs direct endomembrane trafficking in plants, current Opinion in Plant Biology, 2017, 40:22-23

Mayers at al., SCD1 and SCD2 form a complex that functions with the exocyst and RabE1 in exocytosis and cytokinesis, The Plant Cell, 2017, 29: 2610-2625

Camacho et al., Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase, Journal of Cell Science, 2009, 122: 4383-4392

Work in progress

Plant & zo

The science of plants and more


Work in progress

This last week I have been busy with organising my results and preparing them for a presentation that I gave for some of our department. And although I have made some progress with my project since I started it, the presentation I gave highlighted the lack of useful progress during the last half year or so. I say useful progress on purpose. Of course you always make some progress, if only by figuring out why something did not work. But my last year has been basically that, finding out why something did not work in my protein purification procedure, amend this, just to have something else going wrong.

So what I try to do is purify proteins that bind to a phosphoinositol lipid. These phosphoinositol lipids come in different flavours which have both a specific shape and a charge. This enables proteins that bind those phosphoinositol lipids to discriminate between them. Although, you have proteins that bind depending both shape and charge, specific binders, however, others will bind based on charge only, unspecific binders. In my project we are primarily interested in those specific binders. In order to determine which proteins bind specifically and which unspecifically we like a step in the purification procedure that would allow us to discriminate between them. And it is step that primarily gives me trouble. Sometimes it works, but then at other times it doesn’t. As if having optimised the binding to the phosphoinositol-beads affects the ability to discriminate between specific and unspecific binding proteins.

Different floavours of PIs (2)
Different flavours of phosphoinositols

 

The good thing that came out of my talk was that when another PI suggested to do the purification without the step to discriminate between specific and unspecific binders, in the end my PI gave in. I now will be faced with the larger task afterwards to weed out the unspecific from the specific binders after having identified the proteins from the purification. But for now I am excited to finally be able to do the big scale purification for the protein identification to get that list of phosphoinositide binders to work with.

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