Not just grease: packing property of cholesterol powers dynein motor proteins

Cholesterol in vesicle membrane helps dynein power cargo trafficking to the lysosome

 

Short Summary

Inside the cell, cargo-containing vesicles move on microtubules (MTs), powered by protein motors called kinesins and dyneins. Motor proteins work uni-directionally: dyneins transport vesicles inwards {into the cell (minus end of MTs)}, while kinesins move vesicles outwards, in the opposite direction.

This paper makes two points about inward vesicular trafficking: (1) that cholesterol-enriched domains (lipid rafts) help drive dynein motor clustering, a mechanism that helps dynein generate sufficient force for a one-way trip of a late phagasome to the lysosome and (2) that Leishmania donovani (causative agent of kala azhar), a successful intracellular pathogen, secretes lipids that disrupts lipid rafts, thus affecting dynein function and stonewalling the transport of the vesicle that contains it.

Phago-lysosomal fusion

Intracellular pathogens are ingested into vesicles called early endosomes that mature, by changing protein and lipid content, into late phagosomes. These finally fuse with the lysosome, where the pathogen can be degraded. Early endosomes have both dyneins and kinesins, which pull in opposite directions, like a tug-of-war, so they move in a sclerotic manner. But as the vesicle matures, kinesins are lost and dyneins are enriched on its surface. This propels the vesicle in one direction.

How do dynein molecules accomplish this from a physical point of view? The force generated by a single dynein molecule is ~1.1pN, while a late phagosome requires high force (~16pN) to move.

The physics angle

One obvious way to generate more force is to add more dyneins, which these vesicles do, two at a time. However, just more is not enough. Given that vesicles are spherical, a homogenous distribution of dynein molecules will not result in sufficient number being available to engage the MT and vesicle. To increase the area of contact between the MT and vesicle, dynein molecules have to be clustered. This paper provides the first evidence that on late phagosomes, dynein is clustered and this clustering is promoted by cholesterol-enriched lipid rafts (Figure 3). This anchoring of dynein molecules is facilitated by Rab7, a membrane-associated protein enriched on late phagosomes.

How is this information useful?

Besides the advance in fundamental knowledge, that lipids can influence motor proteins, and thus vesicular trafficking, this paper goes on to explore how pathogens exploit this mechanism. Leishmania is an intracellular protozoan pathogen: to stall phago-lysosomal fusion, it secretes lipids called lipophosphoglycans (LPG), that disrupts the raft-like clustering of lipids on late phagosomes. This allows it to subvert transport to the lysosome and survive as a pathogen inside the cell.

Experimental technique

This paper uses a microscopy technique called optical trap. As with experimental science, the first two figures in the paper are dedicated to demonstrating that the technique works, and how its data can be utilised. First, a latex bead is fed to a cell. At different time points, the bead, which the cell tries to transport towards the lysosome is recovered. Early on (<10mins), the bead is an early endosome (EE), while >30mins later it is a late phagosome (LP). The beads are trapped optically and the force that it uses to escape the trap is inferred as the force being applied by the dynein molecules. The force measurements are in the order of pN (10-12), allowing precise measurements of very small magnitudes. Measurements from beads are compared to EEs and LPs purified from cells to prove that the in vitro construction reflects what normally happens in a cell. The beads can thus act as a convenient <case study>. From beads, the general observation emerges that LPs have different transport properties than EEs. Fluorescence images reveals that on LPs, but not EEs, dynein cluster and that these clusters are enriched in cholesterol, and proteins that like rafts.

Take Home

Phagosome maturation and fusion with lysosome is critical for clearance of unwanted material from our cells. This process requires dynein-driven phagasome movement to the minus end of the microtubule. The requisite force for unidirectional travel is provided by clustering of dynein, which is underpinned by clustering of lipids by cholesterol. Pathogens try to escape the clearance mechanism by making lipids that disrupt cholesterol-clustering, and thus dynein clustering.

Reference

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes. Rai A, Pathak D, Thakur S , Singh S, Dubey AK, Mallik R.

An interview with Roop Mallik

Q. The cartoon model of phagasome movement is a ball moving along a stick. However, from a molecular point, is the phagosome tumbling or rolling like a drum i.e., moving as each cholesterol-enriched dynein cluster grasps the MT, or is it walking, the cluster assembling and disassembling rapidly to move along the MT?

Thank you for asking that question. I don’t think that this phagosome is actually rolling along the microtubule. What happens is that once it is in the proper geometrical orientation where a certain group of these motors is close to the microtubule and the motors attach, that will prevent the rolling motion and from our calculations, as you can see in the paper, we think that on this entire surface of the phagosome there are roughly three such clusters, so the average distance between these clusters is likely to be quite large, I mean the angle between them is likely to be quite large. So just by rolling in this manner, going past one of these clusters and finding another cluster, that chance would be fairly low, so I think like most of the time it is on one cluster and then the thing detaches and if it reattaches again to another microtubule it might be on a different cluster. I have no way of proving this but simple physical arguments suggest this kind of a pitch.

Q. Do you think that the mechanism of cholesterol-enriched membrane domains modulating dynein, would be conserved in non-phagocytic cells, like neurons?

Yes, I think so, I don’t have direct evidence for this but the reason is that many of the marker proteins that come on to these phagosomes are pretty much the same whether they are phagocytic cells or non-phagocytic cells. In my view, the only difference is that the cells which are “professional” phagtocytes, their phagocytic rates are much higher and they phagocytose much more and probably the entire process of phagosome maturation is accelerated (is faster) in the professional phagocyte cells. In other generic cells like Cos7 or something like that which are not phagocytic cells, fewer things would be phagocytosed and probably their maturation process would be slower but essentially if you slow down the whole process the sequence of events I don’t think it would be very different but I have not studied this in non-phagocytic cells, so this is just my guess.

Q. Would all cholesterol-enriched domains with Rab7 bind dynein or do you foresee other molecular players that sequester dyenein specifically to Rab7-positive late phagosomes?

There are certainly other molecular players; cholesterol is just probably the scaffold on which things assemble. What we think is that there is a series of proteins via which dynein attaches to these cholesterol-rich domains and I don’t want to take too many names but at least there are proteins which have GPI anchors which specifically bind to cholesterol-rich domains and one of such proteins is a protein called ORP1L, which in turn recruits downstream proteins like Rab7, RILP and then comes dynein and then actin. Interestingly Rab7, which you mention in your question, also has a GPI anchor and we suspect that it plays something like a Picket fence type of role because the Rab7 protein embeds its GPI-anchor probably into the cholesterol-rich domain and then once that happens, the Rab7 also has a property to dimerise. So if you now think of two fences embedded in the membrane and these two poles want to come together, that would stabilize this entire structure right because we embed something and bring them closer that would create stability in the structure. Those kind of models have been discussed in the context of other lipid microdomain-associated proteins, also Rab7, but as you understand these are not things which are easy to visualise most of the biochemical studies which reach these conclusions may have certain artefacts. So that’s where we are right now.

Q. The Leishmania data suggests that pathogens may actively exclude cholesterol from late phagasomes to prevent being trafficked to the lysosome: low cholesterol means slower trafficking. Therefore, would people who have naturally low levels of cholesterol be more susceptible to infections?

That is a interesting question. There is no data for this but all I can tell you is that the Leishmania parasite actively down regulates cholesterol metabolism when it infects you by targeting a specific microRNA so it is a possibility, but this is something which would be interesting to find out whether such co-relative studies have been done. But if you think about it mostly Leishmania is endemic to rural regions where affluent people do not live. So certainly those people are not going to be eating the kind of diet which gives you too much cholesterol. I guess that’s the best I can do right now.

Q. What was the motivation behind studying Leishmania, over other intracellular pathogens like Salmonella or Mycobacterium?

Right so that is was partly motivation and it was partly whatever you can get your hands on. So we had this reagent, lipophosphoglycan which some people were kind to give to us and we did not have any equivalent eeagent for lets say, mycobacteria or Salmonella. Now that is one part of the reason, the other reason why we chose Leishmania is because there are already published papers from the group of Michel Desjardins which showed that a specific protein, flotilin binds to cholesterol-rich domains; they could show that this clustering is disrupted in Leishmania. So we suspected that Leishmania somehow probably by the use of this lipophosphoglycan maybe disrupting this cholesterol cluster. What we were bringing in new into this picture was the presence of dynein in these cholesterol clusters so the possibility that Leishmania could disrupt these cholesterol clusters is not directly our finding, it is somebody else’s but implication in terms of transport is something which we believe we have contributed

Q. You trained as a physicist, but moved into biology. What inspired this? What would you advice those students who are contemplating such a switch?

I will answer that in two parts. What inspired me, one is that I got to know my present wife who was a biologist around the time I transitioned into this and secondly I would say that I really enjoyed what I did for my PhD, but I was looking for newer things at that time and I am not a person who can plan for months and years in advance; this was something that looked interesting at the point of time and I just took to it and things just worked out. It was not part of a great vision or a plan. I will just be honest with you. And then your second part is that what advice would I give to physicists who would want to come into biology. So in my experience and this is my personal opinion when I talk to physicists who want to do Biology, their idea of doing Biology is going and measuring something and putting some number onto something. That is good to begin with but I think that’s not very useful in the long run. To understand the context of the problem and to kind of realise what is going to be important to Biology, to the body and kind of contextualizing that is more important than putting numbers onto things and as a physicist when you study a biological problem and putting numbers to something is not the greatest thing that you can do. Try to give it your own idea of how things evolve in space and time that is something which I think physicists can bring to the plate, while many biologists in my opinion often miss that point. They more interested in the molecular interactions you know, this interacts with that, which is all very useful but try to think of it, bring in the element of space and time and sequence of things is something that at least I try to do as a physicist, I don’t know what I am a physicist or a biologist, whatever, but that has helped me in thinking about my science. That’s my view.