Why stem cells silence virus growth?

Retroviruses can integrate their viral genome into the human genomes. Embryonic stem cells are equipped with a mechanism to shut down the replication of retrovirus. But this mechanism is not commonly found. Thus, it is an interesting question to ask why our stem cells have this special silencing mechanism. Stem cells are transcriptionally active, as it gives rise to multiple lineages of cells. Therefore, it is important to silence the retrovirus transcription. Here, we will discuss the first article of the week, “Embryonic stem cells use ZFP809 to silence retroviral DNAs) by Daniel Wolf and Stephen P. Goff (do ic 10.1038/nature 07844).

Inside the Moloney MLV, there is a primer binding site to initiate replication of the MLV genome. This primer binding site binds to proline tRNA. Thus, using proline tRNA, we can detect the silencing factors that act at the primer binding site to stop replication inside stem cells. In the figure on the right, when Pro probe incubates with the stem cell extracts, lane 2 shows the Pro probe binds to a complex at around 45kDa. (The B2 is just a mutant version of Pro that they know is not going to bind to the silencing factor. Thus, it is a negative control). After mass spectroscopy and subsequent protein analysis on the complex, they figured that the complex composes of a factor called ZFP809. In the bottom figure, you see ZFP809 binds to the Pro probe. Then the author moved on to prove that the ZFP809 blocks the replication of Pro-dependent replication. In short, this paper is the first to find a silencing factor in stem cells to inhibit the replication of MLV.

spotlight articles: cytoskeletal control of CD36 diffusion (Jaqaman et al. 2011) review

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.

References:

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.

SPOTLIGHT ARTICLES: Patronin regulates the microtubule network (Goodwin et al. 2010) REVIEW

Microtubule dynamic is tightly regulated by assembly-promoting and destabilizing factors, as it takes an essential role in cellular processes, such as mitosis and intracellular transport.  In this paper, Goodwin and her group hypothesize that Drosophila Patronin has a regulatory role at the microtubule (MT) minus end. This hypothesis is based on a previous genomic screen that associated spindle morphology defect to the loss of Patronin; and the observation of its human homolog at the minus end. The remarkable stability of free MT minus end has been reported, however the mechanism is yet elucidated. As the majority of research has focused on the plus end, this paper is highly significant by identifying Patronin as a protecting factor at the minus end competing against depolymerizing protein, Kinesin-13.

To approach the hypothesis, Goodwin and her group derived two major experimental procedures: (i) photobleaching, and (ii) in vitro assays. First, photobleaching was used to look at the movement of MT. It created a “dark box” on a region on the MT to identify whether the MT is either transported by motor protein or treadmilling. The bleach mark would be stationary if transported, whereas it would move toward the minus end if treadmilling. Furthermore, several in vitro assays were performed. Notably, the anchoring assay was performed by attaching GFP-Patronin to Anti-GFP coated coverslip, and subsequently observing the rhodamine-labeled microtubules from one end. This anchoring assay allowed the group to identify which MT end is anchored to GFP-Patronin. For the gliding assays, kinesin or dynein was added after microtubule anchoring. As kinesin moved toward the plus end and dynein moved toward the minus end, the binding selectivity of Patronin can be determined.

By comparing to RNAi control, the group found that most Patronin-depleted Drosophila S2 cells have a lowered MT density and an increase of free MT during interphase. These free MT were released from nucleating sites and treadmill across cytoplasm to the cell periphery. The group continued the investigation to explain the increased depolymerization.

Image

The codepletion of Kinesin-13 member (shown in figure), KLP10A, and Patronin, reversed the Patronin-depleted phenotype. During metaphase, the codepletion achieved longer pole-to-pole metaphase spindle than control, and decreased poleward flux of tubulin subunits. Interestingly, Patronin-depleted cells displayed two distinct types of bipolar spindle: normal form that aligns with metaphase plate; and collapsed form that resemble monopolar spindle. From the domain analysis of Patronin, the CC domain was localized with small MT nucleating foci, and the CKK domain was localized along MT. From the gliding assays, Patronin was found to bind to MT at the minus end. In the presenceof Patronin and Kinesin-13, Patronin, MT depolymerization was only shown at the plus end but not at the minus end. Increasing KLP10A homolog, MCAK, also correlated with higher MT depolymerization at minus end when the concentration of Patronin remains constant. In conclusion, Patronin protected the MT minus end against Kinesin-13-mediated depolymerization.

Goodwin and her group have presented a convincing story of the protective function of Patronin. Different domains of Patronin are localized in distinctive regions, suggesting domains might work cooperatively. CC domain of EB1 is necessary for EB1 to bind to MT. Here, CKK alone is able to direct Patronin to localize along MT. Why is there a redundant role of two different domains in localizing Patronin to MT?  The CKK and CH domains may be required to target to the minus end. The CKK and CH domains may need to interact to uncover buried residues for the minus end targeting. To approach the hypothesis, we should align different Patronin homologs. Then, screen out the potential conserved regions in CKK and CH domains. Next, mutate charged residue to alanine residue. If the region is the sites required for minus end specificity, alanine mutants should not localize to the minus end of MT. These results would help us to understand how Patronin obtains its MT minus end specificity.

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Reference:

Goodwin SS, Vale RD. Patronin regulates the microtubule network by protecting microtubule minus ends. Cell. 2010 October 15; 143(2): 263-274.