Inside each individual cell, there are a lot of things need to get transported. Factors that are responsible for DNA replication need to get inside the nucleus. Waste also needs to be removed from the cell interior. The bulk of the transportation system in the cell is supported by the network of cytoskeleton. Proteins can be pulled from point A to point B via the cytoskeleton. In other words, the cytoskeleton works in a similar fashion as an escalator: taking passengers(proteins) to their destination. In the figure on the left, you can see the green structure. That is the cytoskeleton. It stretches from the blue stained structure (which is the nucleus) to the outskirt of the cell (ie. the plasma membrane). To date, there is a model on how cytoskeleton restricts the movement of proteins at the plasma membrane. This model is called the diffusion barrier model, in which the cytoskeleton forms grids (see figure below) restricting the movement of proteins within this grid. The following is my review on a recent article supporting this model.
Cytoskeletal diffusion barrier model attempts to explain the restricted mobility of proteins within the lipid bilayer using the fence concept. In this paper, Jaqaman and Kuwata hypothesized that the clustering of CD36 receptor is regulated by the cortical actin filaments and microtubules. The fluid mosaic model assumes that proteins can move freely within the 2-dimensional bilayer, however, based on observation, proteins are translocated in a somewhat restricted manner. Their finding extends the paradigm that the protein is in fact moving within a 1-D plane defined by cytoskeletal fences. Their paper is, therefore, remarkable as it provides strong evidence supporting the cytoskeletal diffusion barrier model. (reference: Cytoskeletal Control of CD36 Diffusion Promotes Its Receptor and Signaling Function)
To approach the hypothesis, they utilized three major techniques: (i) single particle tracking; (ii) in vitro assays; and (iii) trajectory analysis. First, anti-CD36 antibody was added to primary human macrophages, followed by detection with a secondary Cy3-conjugated antibody. This allowed the group to image a single receptor, and to photobleach a particular receptor to track the motion of CD36 receptor. Next, transferrin and oxLDL uptake assays were performed, in which fluorescent-labeled transferrin and oxLDL was incubated with macrophages to look at the internalization of ligands under various drug treatments: latrunculin B to depolymerize F-actin; blebbistatin to inhibit actin motor, myosin II; and nocodazole to depolymerize microtubule (MT). Furthermore, the trajectory of CD36 receptors was characterized by two methods: scatter of receptor positions; and mobility and displacement of the receptor. The mobility and the displacement were determined by a moment scaling spectrum, in which it plots scaling coefficients against moment order. This plot allowed the group to determine whether the motion is diffusion or not by simply evaluating the slope of the plot.
The group found that the majority of CD36 receptors is surface bound. On the surface of macrophages, they exhibited different types of trajectory: 27% in linear trajectories (LT); 18% in isotropic unconfined diffusion; and 55% in isotropic confined diffusion. These receptors also underwent fusing, splitting, and in relatively rare cases, clustering. LT-CD36 receptors had higher gradient in particle intensities and higher probability of merging and splitting comparing to other trajectory types, indicating that the linear motion is favored in metastable clustering. LT-CD36 also exhibited features that resemble diffusion-dependent process, such as 1D random walks, unchanged frame rate, receptor perpendicular displacement and restriction of its walk with a bias toward the edges. Therefore, the linear motion of CD36 receptors was driven by diffusion. The group continued to study how cytoskeletal perturbation affects the clustering of receptors and the downstream pathway. Depolymerization of MT, F-actin and inhibition of myosin II in macrophage had an adverse effect: decreased linear diffusion and clustering of CD36 receptors; decreased receptor density on cell surface; and decreased CD36 ligand, oxLDL, internalization. Furthermore, inhibition of myosin II and depolymerization of MT suppressed downstream c-Jun phosphorylation. In conclusion, cortical actomysoin and MT network are both important in arranging the linear motion, clustering and downstream signaling of CD36 receptors.
Jaqaman and Kuwata had presented a convincing story. The control of cytoskeleton on signaling pathway however requires more support. phos-cJun assay is dependent on the fluorescence output to quantify JNK activation. As several 2° antibodies may be bound to a 1° antibody, the assay may not be accurate. I suggest the group to quantify the phos-cJun by Western blotting. Future direction should focus on whether the geometry of the ‘fence’ would affect the signaling pathway. This would be important assuming that the bilayer is dynamic and all fences are not the same size. To approach the question, we can design an experiment in which we overexpress crosslinkers in cells(results in thicker actin and decreased 1D fenced space). Then look at the walk of Cd36 receptor (decreased space should result in restricted walk). From here, we are closer in demystifying the factors behind the ‘random’ walks of proteins.
Cytoskeletal Control of CD36 Diffusion Promotes Its Receptor and Signaling Function Original Research Article
Cell, Volume 146, Issue 4, 19 August 2011, Pages 593-606 Khuloud Jaqaman, Hirotaka Kuwata, Nicolas Touret, Richard Collins, William S. Trimble, Gaudenz Danuser, Sergio Grinstein. Cell. 2011 Aug 19;146(4):593-606.