Gene expression ≠ protein accumulation

Gene expression ≠ protein accumulation

I was remined lately that people forget that gene expression does not equal protein accumulation. And also that protein accumulated is not equal to protein that is actual able to do what it is made for. So here an explaination about gene expression, protein accumulation, why are they not the same, and why protein accumulation is not telling anything about functionality of the protein.

Gene expression

As I explained to my mum last night, you can think about it like this: when playing with LEGO, if you are making one type of car over and over again you can measure how fast you assemble that car. This measurement of assembly we call expression, so in this case it is LEGO-car expression we measure, for the expression of genes it is called gene expression.

Protein accumulation

Expression is however is telling you nothing about the number of cars of that type of car are actually there. That is not only dependent on the speed of car assembly (LEGO-car expression), but also on the speed of car disassembly. If assembly and disassembly are in balance it does not matter if the assembly speed (LEGO-car expression) is high or low because the total number available cars does not change. The total number of available we call accumulation, for our LEGO-cars it is LEGO-car accumulation, when we talk about the total number of available proteins it is protein accumulation.

Protein functionality

This explains how protein accumulation is dependent on more than just gene expression. In turn protein functionality is dependent on more than just protein accumulation. It is also dependent on something called posttranscriptional modifications. Back to our cars. If you have 5 vans that you use to deliver goods, but three are stuck at the petrol station waiting for fuel, then effectively you have only 2 vans that are able to go on their delivery rounds. In this example you can see being stuck at a petrol station as a posttranscriptional modification that takes away the functionality of the protein (3 vans are not functional) without changing to overall car accumulation (there are still 5 vans). The simple measure of supplying fuel (posttranscriptional modification) will increase the number of functional proteins without changing the overall number of vans (car accumulation).

Hopefully this will help you remember that gene expression does not equal protein accumulation and that protein functionality is dependent on more that just protein accumulation. No just remember, protein does not equal car.

PLC sandwich

PLC sandwich

Another genius bit of biological design comes from the enzyme PLC. PLC is a phospholipase whose job it is to cleave of the headgroup of PI(4,5)P2, a membrane lipid. Like tethering proteins, PLCs are made up out of a couple of domains. At its core there are three domains. There is the catalytic domain, which is doing all the action. Then there is a lipid binding domain, C2 domain, whose job it is to attach the protein to the membrane, preferably near PI(4,5)P2. Last there is a EF-hand domain, which likes to interact with other proteins. The order of these domains in the proteins is as follow:  EF-hand domain – catalytic domain – C2 domain. Mammalian PLCs may have a couple of other domains, so called regulatory domains, on either end. But plant PLCs don’t so it is unknown how exactly their activity is regulated.

chematics PLC
A scematic overview of the core of PLC proteins.

However, there have been some studies done on which domains are required to be there for which action, and how the protein looks when it is all folded up. First its structure, in contrast to its sequence order, when folded up the C2 domain sits in the middle. With on one side the catalytic domain, to which it is loosely attracted to. On the other side is the EF-hand domain to which it feels quite some attraction so it huddles close to it. By doing so it stretches the linker between the EF-hand domain and the catalytic domain, shoving the catalytic domain close to the C2 domain. In doing so, when bound to the membrane, it places the catalytic domain just right so it can grab hold of a PI(4,5)P2 headgroup to cleave it off.

scematics structure PLC
A schematic overview of how the core of the PLC protein looks when folded up, with the EF-hands binding to the C2 domain, forcing the C2 domain and catalytic domain close

This by itself is just brilliant already. Looking at what is required for what function, expectantly they found that for PLCs membrane localization its C2 domain was just enough. However, looking at what is needed to chop of the headgroup of PI(4,5)P2 things became puzzled. It turned out that with just the catalytic domain, nothing happens, likely because the membrane could not be found. But having the catalytic and the C2 domain is not enough either. The EF-hand domain needs to be there, holding them all close together, so the catalytic domain can find PI(4,5)P2.

This suggests that when the EF-hand domain is not bound to the C2 domain, the catalytic domain is just hanging of the C2 domain, away from the membrane, unable to do its job. Implying that controlling the interaction between the C2-domain and the EF-hand domains is a way of regulating PLC’s activity.

Realizing this was quite an eyeopener for me. However, what surprised me more was that although this was all discovered over 15 years ago. No subsequent research seem to have been done to find out the details of how the interaction between the C2 domain and EF-hand domain is regulated.

Literature

Essen LO, Perisic O, Cheung R, Katan M, Williams RL. Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta. Nature. 1996;380(6575):595-602. doi:10.1038/380595a0

Otterhag L, Sommarin M, Pical C. N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana. FEBS Lett. 2001;497(2-3):165-170. doi:10.1016/s0014-5793(01)02453-x

Jiménez JL, Smith GR, Contreras-Moreira B, et al. Functional recycling of C2 domains throughout evolution: a comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches. J Mol Biol. 2003;333(3):621-639. doi:10.1016/j.jmb.2003.08.052

Tethered to science

Tethered to science

For a long while I haven’t posted anything, being too tired and work got in the way. Then after combing back from a conference and a break late February, full of ideas but no energy to work them out, I turned to my GP.  There I was told I was overworked, warned that if I did not slow down I will end up with a burn out. So I tried to rest and slow down, while still having that nudge of guild when leaving work early. Then the corona crisis hit. Forced to work at home made me feel less guilty for the times I stopped earlier, because hey we where in a crisis, so its ok not to be able to concentrate. Needless to say it did wonders for my recovery.

Working on a review article that was already in the pipeline, I recovered slowly. Not only am I again able to work a full day without loosing my concentration. I also found back what I lost long ago. How much I enjoyed just reading articles, being able to follow up on what I read, while discovering how it all worked. Then finding a way to describe this so others would see the connections between the different studies as well.

In short it showed me that biology is amazing and has find some inventive solutions to its problems.

One of these are tethering proteins. These are proteins that are connected to membranes of different organelles, say the endoplasmic reticulum (ER) and the plasma membrane, to keep them close to each other. Tethering proteins make use of two different domains. On one end of the protein they have a transmembrane domain. This domain insert itself through the membrane to anchor the protein to it. On the other end they have a couple of domains that can bind membranes.

Tethering protein
A schematic overview of a tethering protein, TM: transmembrane domain, C2: C2 domain.

There are lots of different types of domains that can do that. But there is one in particular that is quite interesting in this context, the C2 domain. The C2 domain binds to membranes in a calcium dependent manner. But the concentration of calcium required depends on the exact sequence, so there are C2 domains that need just a little bit of calcium and they will already bind membranes. Then there are others that need quite a high calcium concentration for them even have a chance to bind the membrane. Tethering proteins make use of these C2 domain characteristics. They have a low calcium requirement C2 domain at the extreme end of the protein, and then another, one, two or three C2 domains, each requiring a bit more calcium before they are able to bind the membrane, placed more towards the transmembrane domain.

tethered membrane low Ca2+
A tethering protein holding two membranes together under low calcium conditions

In this way, by using tethering proteins, the cell can hold both, say the ER and the plasma membrane, just having a little bit of calcium present.

Tethered membrane high Ca2+
A tethering protein holding two membranes together under high calcium conditions

But when the ER and the plasma membrane needs to be really close, say for some signalling function, then the cell can increase the calcium concentration, and the tethering protein will just reel in the ER close to the plasma membrane.

That image of the ER tethered via a leach to the plasma membrane, so it can be brought in close when needed. Is just one of the many I got over the past weeks, reading for my review. I will share some more over the coming weeks, illustrating how science reeled me in once again.

Literature

Brault ML, Petit JD, Immel F, Nicolas WJ, Glavier M, Brocard L, Gaston A, Fouche M, Hawkins TJ, Crowet J-M, et al (2019) Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata. EMBO Reports 20: e47182

Ishikawa, K., Tamura, K., Fukao, Y. and Shimada, T. (2020), Structural and functional relationships between plasmodesmata and plant endoplasmic reticulum–plasma membrane contact sites consisting of three synaptotagmins. New Phytol, 226: 798-808.

Jiménez JL, Smith GR, Contreras-Moreira B, et al. Functional recycling of C2 domains throughout evolution: a comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches. Journal of Molecular Biology, 333(3):621-639.

Plasticity in phloem development

Plasticity in phloem development

Last week at a symposium, we were reminded by Antia Rodriguez-Villalon that in plants organogenesis does not stop after germination. In fact, plants keep producing new organs through their lives. While most of us think by organ formation in plants first about leaves or flowers, Antia Rodriguez-Villalon work actually focusses on vascular development in roots. Her main take home message was that vascular development is more plastic than we initial thought. And that this plasticity safeguards the development of a functional vascular system. So I was excited when this weekend I saw in a tweet about the latest article of her group describing this study.

protophloem - root
The radial organization of the vascular tissues in Arabidopsis roots where depicted phloem tissues are color coded (CC, companion cell; MSE, metaphloem sieve element; PPP, protophloem pole pericycle cells; PSE, protophloem sieve element). Copied from Gujas et al. 2019

Before I go into more details about her work we will take a short detour, about phloem development. In plants the fate of a cell is determined by its position. As such, we know the function of a cell by its position in the plant. In Arabidopsis roots the phloem pattern is made up of four cell types, and is well conserved. With the protophloem and the metaphloem sieve elements originating from a common stem cell, whereas the companion cell originates from a different stem cell. In effect, once the precursor cells to the protophloem and the companion cells get pushed outside the meristem region, these cells are in different but adjacent cell files. Cells that have a protophloem identity can be visualised with a protophloem marker.

Using this technique they looked at how the protophloem identity was affected in the mutant, cvp2 cvl1, which is severely compromised in protophloem development. Finding that protophloem identity was affected, they set out to determine the new identity of these affected cells. Surprisingly, these affected cells had a gene expression similar to that of companion cells. Investigating this a little further, by intentionally disrupting the protophloem development, showed that in case of disruption the protophloem identity switched from the protophloem cell-file to the companion cell cell-file. Giving the first hint of the plasticity of the protophloem development. This plasticity is restricted to a so called “plastic zone” in which the cells surrounded the protophloem are still in an uncommitted stage. Eventually, through growth, these uncommitted cells will be pushed out of the plastic zone and commit.

Further study to investigate how the plastic zone is regulated identified that RPK2 and CLE45 function to restrict protophloem identity to the protophloem position. In addition, CLE45 treatments prevented the plasticity of protophloem development. Suggesting that in the plastic zone, plants normally can modulate CLE45 perception at single cell level, enabling them to re-pattern to form a functional phloem pole upon positional cues.

protophloem plastic zone schematics
Schematic Overview of the Molecular Mechanisms Regulating PSE Identity and Phloem Patterning in Arabidopsis Roots. A longitudinal view (left) and radial view (middle) of the developmental trajectories of protophloem sieve elements (PSEs) and companion cells (CCs) within the root are represented. Previous to their entry into the plastic zone (surrounded in red), future PSE cells acquire their cell identity and enter into a proliferative phase, a process partially regulated by the activity of positive regulators (such as CVP2) and counteracted by negative regulators, such as CLE peptides. Within the plastic zone, PSE-surrounding elements are primed as phloem cells, but they still exhibit plastic identity and can switch their identity (red arrows) according to positional cues. Once PSE cells enter into differentiation process, RPK2 excludes PSE identity from PSE-surrounding cells, allowing these cells to commit to CC’s developmental trajectory. Copied from Gujas et al. 2019

This latest work of the group of Antia Rodriguez-Villalon showed us that phloem development has a back up plan for it worse case scenario. I look forward for them to find out more about how this is organised.

Literature

Gujas et al., A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements, Current Biology (2019), https://doi.org/10.1016/j.cub.2019.12.043