|Phospholipid undergoes hop diffusion in compartmentalized plasma membrane.
This slide show has been produced based on talks that Dr. Kusumi gave on several different occasions. Therefore, it is written in a lecture/seminar style.
The most relevant publication is
In this slide show, we deal with compartmentalization of the plasma membrane that even acts on diffusion of phospholipids in the plasma membrane (therefore almost any molecules in the membrane). Such knowledge forms the basis for understanding molecular interactions and recruitment occurring in the cell membrane.
This is a typical trajectory of a transmembrane protein called CD44. This particular molecule moved about rapidly, but was confined in a region of about 600 nm for a few seconds, and then hopped to an adjacent compartment, and then again started confined but fast diffusion, and hopped to the next, and so on. We have examined various membrane proteins and carried out quantitative and statistical analysis, which I don't have time to explain to you, but we found that many membrane proteins undergo macroscopic diffusion over many compartments by repeating such confinement plus hop movements, which we call hop diffusion.
These lines here are to help the eye, but we showed that, in fact, these compartment boundaries are made from the membrane skeleton.
The transmembrane protein can cross the barrier when the membrane skeleton or the membrane itself fluctuates to give space between the membrane and the membrane skeleton, or when the membrane skeleton temporarily dissociates or when the transmembrane protein has kinetic energy sufficiently high to get over the membrane skeleton fence barrier when it is in the barrier region.
Membrane biophysicists have been puzzled for these 25 years by the observations that diffusion rates of membrane proteins in the plasma membrane are reduced by a factor of 10 to 100 from those in artificial reconstituted membranes, and we thought we largely solved this puzzle with this membrane skeleton fence model. Namely, diffusion in the plasma membrane is slowed not because the diffusion rate per se is small, but because transmembrane proteins are corralled by the membrane skeleton mesh.
I said "we thought" using the past tense, and this will be clarified toward the end of this Slide Show 2.
If you have already looked at the Slide Show 1, please skip the Suplementary slides. They are the same as those shown there, but are reproduced here for those who have not seen Slide Show 1.
One of the critical issues with the membrane skeleton fence model is whether or not membrane proteins actually collide with the membrane skeleton. To examine this issue, we carried out the following experiment using red blood cells.
We attached a large bead to the membrane skeleton. At the same time, we labeled a transmembrane protein band 3 with small colloidal gold particles. And we only observed the band 3 that are undergoing hop diffusion.
Then the membrane skeleton was deformed like this by dragging the beads using laser tweezers. If band 3 molecules collide with the membrane skeleton, they will be moved along with the membrane skeleton as shown. If band 3 molecules are not colliding with the membrane skeleton, their movements will not be affected by such displacement of the membrane skeleton.
So, let's look at the result in the next video sequence.
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Next, I would like to show you that not only proteins but also lipids are corralled by the MSK. This occurs due to the effects of transmembrane proteins that are anchored to the membrane skeleton, and we call these anchored proteins pickets, because they act as posts for the membrane skeleton fence.
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Next is a gold tagged DOPE. This is in real time and recorded at the video rate, and the scale bar shows 1 micron. As you can see, gold-tagged DOPE appears to diffuse as fast as Cy3-DOPE.
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However, achieving higher time resolutions in single fluorophore observations is difficult due to the problem of poor S/N ratios. Therefore, we had to depend on gold-tagged DOPE and single particle tracking. Using a high-speed video system and a modified microscope, we were able to achieve a temporal resolution of 25 μs.
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A quantitative analysis showed that the compartment size was 230 nm on average. The residency time within each 230-nm compartment was 11 ms. No wonder we did not see hop movement at the video rate, whose time resolution is only 33 ms. In fact, these trajectories are 62-millisecond long, and if we had used video rate observation, there would only be 2 or 3 points in the whole trajectory.
The diffusion rate within the 230-nm compartment is interesting, which is 5.4 mm2/s on average. It is as fast as DOPE molecules observed in artificial membranes, such as giant liposomes. Therefore, lipid diffusion in the cell membrane is slow not because the diffusion per se is slow, but because the plasma membrane is compartmentalized with regard to the translational diffusion of phospholipid.
We also observed DOPE diffusion on blebs on the cell surface. The bleb is a balloon-like structure of the cell membrane where the membrane skeleton is largely lost, and we found that DOPE was undergoing very rapid simple Brownian diffusion rather than hop diffusion on the blebs, indicating again that the membrane skeleton may be responsible for the hop diffusion of DOPE.
However, this is very strange because the DOPE we are looking at is located in the outer leaf of the membrane, whereas the membrane skeleton is located on the cytoplasmic surface of the membrane.
In this model, transmembrane proteins that are anchored to the membrane skeleton are coupling the membrane skeleton, which is located on the cytoplasmic surface of the membrane, to the phospholipids that are located in the outer leaflet of the membrane.
These anchored-protein pickets would act on any molecule incorporated in the membrane. Therefore, in the case of transmembrane proteins, both membrane-skeleton corrals and anchored-protein pickets are responsible for slowing of their diffusion in the plasma membrane. This is the reason why I used the past tense in the previous part.
In the case of NRK cells, we found a second compartmentalization: the cell membrane has double [nested] compartmentalization (cf. T. Fujiwara, K. Ritchie, H. Murakoshi, K. Jacobson, and A. Kusumi. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 1071-1081 (2002)).
Compartmentalization in the plasma membrane in other cells has been investigated in our laboratory. A paper on this subject is under preparation.
A possibility I would like to suggest is that these diffusion barriers are important for maintaining the spatial information when chemoattractant or chemorepellant molecules are received by respective receptor molecules. For example, when an extracellular signal is bound to a receptor monomer in the center, the receptor molecules tend to form oligomers and/or cytoplasmic signaling molecules are often recruited to the liganded receptor molecules. This would induce a dramatic decrease in the diffusion rates of such signaling complexes, due to the enhanced effects of corralling or binding by to the pickets and fences, and these signaling complexes would immediately be arrested within the compartment where the signal was received. Namely, monomer molecules tend to undergo hop diffusion with relative ease, whereas oligomerized receptor molecules and those bound by cytoplasmic signaling molecules cannot cross the pickets and fences very easily (right), and also tend to be tethered to the membrane skeleton (left).
You might intuitively assume that the diffusion rates of molecules in the membrane would decrease if they form oligomers or larger molecular complexes. However, this is not true. Since two-dimensional diffusion is very very insensitive to the diffusant size, even if the receptor molecules form 10-mers, the diffusion rate will decrease only by less than 20%. Without the pickets and fences, oligomerization-induced or signaling-molecule-binding-induced decrease of motion or immobilization would not occur. Such an arrest could be particularly important for the receptors involved in chemotaxis, the cytoskeletal reorganization, or protrusion of processes, where the receptors have to "memorize" where the signal was received.
We have previously shown that oligomerization of E-cadherin-GFP greatly decreased its diffusion rate, and explained this by an "oligomerization-induced trapping model", which forms the basis of the above argument
This is the end of Slide Show 2. Thank you very much for your interest in this work.