Category Archives: Articles Review

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.

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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.

SPOTLIGHT ARTICLES: Control of allosteric signaling switch (Dueber et al. 2003) REVIEW

Today I will briefly discuss one of the landmark papers in protein dynamics. This paper is called Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination, by John Dueber and Wendell Lim from UCSF, published in Science in 2003.

Allosteric signaling switch has been a focus in synthetic biology, as proteins that belong to the signaling pathways are often regulated by allosteric gating mechanisms. Some of these mechanisms are governed by simple binding domains, in which the switch is under autoinhibited state when proper ligand is absent. In this paper, Dueber and his group proposed that combinations of domains could give rise to a diversified number of gating behaviours in response to nonphysiological inputs. This story is highly significant because their results indicate that simple catalytic and interaction domains within a single polypeptide could give rise to different proteins of complex properties. It also advances our knowledge to how proteins of complex functions could be derived.

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To approach the hypothesis, Dueber had developed two key major experimental procedures: (i) switch proteins; and (ii) actin polymerization assays: pyrene-labeled and carboxylated polystyrene bead. There were three types of switch proteins developed: single heterologous ligand; chimeric switch; and heterlogous switch. To develop switch proteins, the ligand motifs and the linker region were PCR-amplified from existing plasmids. These proteins were then expressed with an affinity tag (either 6xHis tag or GST tag), followed by purification and tag removal by Ni beads and TEV protease or glutathione agarose resin. Next, actin polymerization assays were performed. In the case of pyrene-labeled actin assays, pyrene-labeled actin was incubated with Arp2/3, switch proteins and ligand proteins to initiate polymerization. Then the rate of actin polymerization could be measured by fluorescene measurement. Next, bead actin polymerization assay was performed. Carboxylated polystyrene beads were coated with GST fusions to input ligands and PDZ ligand. After incubation, the beads were washed and incubated with Xenopus oocyte extractsand rhodamine-labeled actin, followed by microscopic analysis on rhodamine.

Dueber developed a signaling switch gated by single ligand. This switch was consisted of N-WASP output domain, GBD and B-motif. At the autoinhibited state, GBD and B motif interacted with output and Arp2/3 to suppress output to interact with actin filament. To initiate actin polymerization, Cdc42 and PIP2 bind to GBD and B motif respectively to relieve autoinhibition. This model was consistent with the observation that increasing PDZ ligands could activate the switch. For the second designs, they developed AND-gate switches by linking two domains with an output. There were two classes of designs developed: chimeric and heterlogous switches. Chimeric switch design was regulated by PDZ ligand and Cdc42. The modular domains of this particular design were PDZ and GBD domains, in which Cdc42 could disrupt interaction of GBD and output. Heterologous switch was regulated by PDZ and SH3 ligands. The modular domains were composed of PDZ and SH3 domains. Both classes of switches were subdivided into behavioural classes. Out of 34 switches, 2 showed antagonistic gating, 2 showed OR gate behaviour, and 5 showed AND gate behaviour. From analyzing the behaviours, the basic design principle was identified, in which the affinity of autoinhibition was correlated with the basal repression and input sensitivity. This principle was supported by 2 observations: (1) reducing the PDZ ligand-affinity for switch C11, the switch then resembled AND gate; and (2) increasing PDZ ligand affinity turned H15 switch from behaving like OR gate to AND gate. Their results also indicated the importance of linker region to switch behaviour, as the increase of linker region from 5 to 20 residues could increase the sensitivity of inputs. Furthermore, in the case of antagonistic switches, PDZ ligand was found to act as an activator while SH3 increased the basal level of repression. PDZ was responsible for the autoinhibitory interaction with output, and SH3 interaction was responsible for destabilizing the PDZ interaction. In the case of positive integrating switches, both domains worked together to downregulate the output activity. Interestingly, disruption of both was required to activate the output function.

Dueber and his group have effectively addressed the main question. The results from simple binding domains and corresponding ligands had supported the notion that complex signaling circuits could be derived from simple domains, and more importantly, domain recombination could potentially derive proteins with novel regulated functions. For future direction, the geometry of the output domain must be investigated. In order for the output domain to be regulated by other domains and to carry its native function, certain geometry must be preserved. To understand the preservation, X-ray crystallography and mass spectroscopy should be conducted on samples: output domain bound to GBD and output domain bound to actin filament.

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Reference: Dueber JE, Yeh BJ, Chak K, Lim WA. Reprogramming Control of an Allosteric Signaling Switch Through Modular Recombination. Science. 2003 Sep 26;301(5641):1904-8.