1,721,114 research outputs found
Recommended from our members
Molecular stochaticity in mammalian cell signaling: Lipid membrane organization and CaMKII kinetics
Full text linkMolecular processes viewed at the single molecule level are stochastic and living cells are full of stochastic processes. Cellular processes frequently occur with a discrete number of molecules and understanding the stochastic behavior of them is of fundamental importance. Here, I studied physical chemistry of two important molecular species in cells: the lipid and the protein. On the subject of lipids, I studied miscibility phase structure of the live cell membrane. Observations of liquid-liquid miscibility phase transition in ternary mixture membranes with hypothetical existence of heterogeneous membrane domains in mammalian cells caused hypothesis of immiscible domains in live cell membranes. Discussion on the subject is often misleading when the discussion is only focused on the qualitative picture of domain existence, but does not consider the physical principles behind it. The question is where in the phase diagram the living cell membrane is poised. To address this question directly I observed physical parameters of the live cell membrane as a function of temperature and I conclude that the live cell membrane is poised reasonably far from the transition temperature. I also discuss the lack of direct evidence for miscibility phase structures playing an important role in actual signaling and the implication of criticality in membrane reactions.On the subject of protein, I studied kinetics of CaMKII, a major protein involved in hippocampal synaptic plasticity. CaMKII holoenzyme has a complex structure comprising of twelve subunits and as a molecular component in a neuronal signaling network, this complex structure allows the enzyme to carry out complicated functions. Using a recently solved x-ray crystallographic structure of the CaMKII holoenzyme, I have modeled the relationship between docked-extended states equilibrium and the calcium frequency response of CaMKII. Stochastic kinetics simulations show that CaMKII frequency response can be fine-tuned by adjusting the equilibrium constant. I also show for the first time, activation dependent subunit exchange of CaMKII dodecamer using single molecule TIRF microscopy. This strongly supports the hypothesis that the CaMKII dodecamer, with its continuous turnover of subunits, can serve as a form of molecular memory
Recommended from our members
Biophysics of Protein Condensates on Supported Lipid Membranes
No full textBiomolecular condensation has been recognized for the last decade-and-a-half as a ubiquitous modality of high-order molecular organization in living cells. This allows for compartmentalized, altered biochemistry that enables otherwise rare biochemistry. Condensates have been observed with both cytosolic (solution), and membrane-bound (surface) components. Linker for Activation of T Cells (LAT) and Epidermal Growth Factor Receptor (EGFR) are examples of transmembrane proteins that can undergo the latter type of condensation. The phase transition of the cytosol-facing phosphotyrosine tails of both of these proteins has been previously reconstituted on supported lipid bilayers (SLB). This type of phase transition is anabled by multivalent binding with downstream adaptors and signaling enzymes to form an extended, two-dimensional bond network. For LAT and EGFR, the adaptor protein Grb2 and the proline-rich scaffolding domain of the guanine exchange factor SOS, together, can reconstitute condensation. Previous work on these signaling condensates have shown that the condensed phase can directly impact downstream signaling kinetics. Additionally, the valency of crosslinking bonds and the bond association and dissociation kinetics within the condensate critically determine the phase transition kinetics and material properties of the resulting condensate. This dissertation details reconstitution of these condensates on SLBs along with fluorescence microscopy to further explore these faucets of signaling condensates. We find that condensation can be driven solely by binding to Grb2 which can dimerize to crosslink signaling proteins without SOS. This dimer interface can in turn be controlled through a phosphotyrosine on Grb2 itself to regulate condensation. This Grb2-mediated condensate retains its enhancement of Ras activation kinetics through SOS which can now be modulated through phosphorylation of Grb2. We also observe that we can reconstitute simultaneous condensates in solution and on the membrane with the same components. The solution condensates, on contact with the membrane surface decorated with a membrane condensate, can undergo a partial transition from a bulk three dimensional condensate to a surface-bound two dimensional condensate
Recommended from our members
Kinetic Resolution of Ras and Arf Signaling Activation by GEFs on Lipid Membranes and in Live Cells
Full text linkSupported Lipid Bilayers (SLBs) were used in conjunction with live cell measurements to better understand the activation of the small GTPases Ras and Arf by their respective GEFs SOS and ARNO. As membranes are crucial for proper activity of these proteins, membrane-mimic assays were developed to quantitatively measure kinetic activation and diffusion.Ras is a common oncogene that causes over 30% of all human cancers. SOS is one of Ras’s activators that is known to play a role in the determination of cell fate. The kinetics of SOS activation of Ras was explored in four of the following chapters. Firstly, a nanofabricated platform that incorporated liposome reaction chambers was developed to assess the kinetic activity of single SOS molecules with single nucleotide turnover resolution. Secondly, the measurement of SOS motility and localization in live cells contributed to an understanding of SOS regulation through a stable membrane-associated active state, resulting in SOS endocytosis. Thirdly, a small molecule inhibitor was shown to impact SOS binding to Ras in vitro and in vivo. Fourthly, the impact of oncogenic Ras mutations on SOS binding and catalytic rate was measured. These results provide insight in the tight regulation of SOS-mediated Ras activation, and also suggest future directions for rational drug design targeting oncogenic Ras.Arf is a small GTPase in the Ras family that regulates membrane traffic and morphology. In the final chapter, a SLB-based assay for GEF-mediated Arf recruitment studies was developed and diffusion rates for both Arf-GTP and Arf-GDP were measured, demonstrating a well-defined, transient membrane interaction for Arf in the GDP state. Additionally, autoinhibition of the GEF ARNO was confirmed. This yields new insight into the mechanism of Arf signaling initiation. Overall, it is demonstrated that in vitro membrane mimics, in combination with live cell measurements, provide a powerful tool to gain a deeper understanding about the activity and regulation of GTPases
Recommended from our members
Role of Clustering in Determining Spatial Organization at the Immunological Synapse
Full text linkMicron-scale assemblies of molecules are thematic in biology, although their mechanism of formation and exact functional role are oftentimes unknown. The immunological synapse (IS)--the gateway event to the body's initiation of an immune response against infection--is a hallmark example. T cell detection of pathogenic invasion on an antigen-presenting cell leads to the arrangement of receptor-ligand pairs into well-defined concentric zones referred to as supramolecular activation clusters (SMACs). The main signaling molecule, the T cell receptor (TCR), binds its specific foreign peptide-presenting ligand, major histocompatibility complex (pMHC). These complexes form a central cluster in the central SMAC (cSMAC) at the center of the intermembrane junction. Immediately surrounding the cSMAC is the peripheral SMAC (pSMAC), populated by a ring of leukocyte function associated antigen-1 (LFA-1) bound to intercellular adhesion molecule-1 (ICAM-1). In this dissertation, we determine how the final IS pattern emerges from a uniform distribution of receptor-ligand pairs. It is known that the actin cytoskeleton drives the centripetal transport of these proteins, but it is unclear how actin sorts them into their final destinations. We postulate that the large-scale sorting of proteins into the different SMACs is a natural consequence of smaller scale protein sorting into microclusters, which may contain hundreds of molecules. To test this, we increase the LFA-1 cluster size two additional degrees beyond its native state with antibody crosslinkers. We either crosslink LFA-1 directly or indirectly with antibodies against its ICAM-1 ligand, which is presented on a supported membrane with the activating pMHC. Progressively more central localization of LFA-1 proportional to the degree of crosslinking results until LFA-1 occupies the cSMAC with TCR. Based on these results, we propose a sorting mechanism based on frictional protein coupling to actin. In the frictional force coupling model, the extent of radial protein transport by actin is determined by the specific coupling chemistry and the protein cluster size. This model predicts cluster size-based protein sorting across the IS. Using fluorescence fluctuation measurements and a small illumination area, we detect a gradient of LFA-1:ICAM-1 cluster sizes across the pSMAC in the native IS, as predicted by our model. Thus, we demonstrate that the well-regulated event of protein clustering is a critical parameter in regulating spatial patterning in the IS
Recommended from our members
Molecular Signal Processing in T Cell Signaling
No full textT cells behave as an extremely sensitive and selective sensor of antigen molecules. Only a few molecules of cognate antigen surrounded by outnumbering non-cognate self antigens are detected by a T cell, leading to T cell activation and immune responses. This antigen discrimination occurs through a system of molecules and reactions, involving T cell receptor (TCR) and intermediate and effector signaling molecules. How the signal processing through this molecular system provides the exquisite capability of antigen sensing remains unclear. Single-molecule observations of antigen-ligated TCR and associated signaling events have revealed that a single ligated TCR stochastically induces a discrete LAT condensate – a local dense phase of a scaffold protein LAT and associated proteins – in an antigen-dependent manner, implying a crucial role of LAT condensation in antigen discrimination. Here we characterize how LAT condensation and associated molecular events process the signals originating from antigens. In the first part of the study, we ask whether the condensates are functional by characterizing the correlations between LAT condensation and downstream calcium signaling using single-molecule and live-cell imaging. We find that individual LAT condensates are functional for calcium signaling without temporal delays. We further quantitatively analyze the LAT-calcium-NFAT signaling axis and find that calcium signal is additively contributed by individual condensates, and suggestively NFAT activation binarizes it. This signal processing operates the summation and thresholding of condensate counts. Furthermore, the finding of the condensate-calcium linear relation reveals the mechanism of calcium signal dynamics in T cells, which has been questioned for decades. In the second part of the study, we address the question of how the dwell-time-dependent LAT condensation propensity emerges from upstream molecular events, which determines the TCR response to antigens with different binding lifetimes, offering antigen discrimination fidelity. A canonical conception of kinetic proofreading describes such fidelity by sequential reaction steps. We propose that a more general kinetic proofreading scheme should reflect two molecular features: the multiplicity of the signaling domain ITAM on TCR, and LAT condensation behaving as a binary output from TCR. A revised scheme features parallel reactions on single TCR which is integrated to induce the binary output, referred to as multi-threading. We theoretically characterize these schemes and find the advantage of multi-threading that robustly improves the discrimination fidelity, which also describes several experimental antigen discrimination behaviors. Together, critical aspects of the molecular signal processing for antigen sensing by T cells are characterized
Recommended from our members
Spatiotemporal Organization of Signaling Proteins on the Cell Membrane Studied by Spatially Patterned Supported Lipid Bilayers
Full text linkMembrane proteins in the cell are dynamically organized to process the information from the environment and can precisely regulate cell signaling and functions. The spatiotemporal dynamics of proteins can be investigated by means of fluorescence microscopy to elucidate the mechanism of cell signaling. In addition, a fluid lipid bilayer on a flat glass surface with two-dimensional mobility of lipid molecules, called supported lipid bilayer (SLB), has proved a powerful tool to investigate cell-cell interactions and reconstitute membrane protein functions in vitro.The T cell is activated by an antigen presenting cell (APC) when a T cell receptor (TCR) is engaged by a major histocompatibility complex (MHC) on the APC. To precisely regulate T cell activation, the T cell organizes membrane proteins and other signaling molecules into particular spatial organization. We applied spatial patterning technologies to the SLB to probe and modulate spatiotemporal dynamics of the T cell signaling, and investigated its mechanism by fluorescence microscopy. In Chapter 2, the SLB embedded with a regular array of gold nanoparticles (nanodot array) was used to probe T cell receptor (TCR) microclusters on the T cell membrane. This nanodot array probes membrane protein assemblies below the diffraction limit of light in living cells by a mechanical means, which complements super-resolution microscopy. In Chapter 3, the spatiotemporal dynamics of Linker for Activation of T cells (LAT), another important protein in T cell signaling, was investigated by localized stimulation to the T cell using a polymer-patterned SLB. This method effectively separated the sites of T cell activation far apart from each other, and elucidated the LAT dynamics upon T cell activation more clearly than ever.ESCRT proteins play an essential role in membrane budding and scission, and it is suggested that they use membrane curvatures to regulate their functions. In Chapter 4, we made a cover glass with nano-hollows of ~100 nm in depth and ~200 nm in diameter, and investigated the interaction between the SLB on the nano-curvature and ESCRT proteins. Highly selective accumulation of ESCRTs into the part of SLB with nano-curvature was observed, which indicated the ESCRTs sense the artificial nano-curvature just as they do in vivo. This experimental platform opens up possibilities for precise kinetic studies on ESCRTs in vitro
Recommended from our members
Reconstitution and Characterization of Membrane Proximal Signaling Downstream of the T Cell Receptor
No full textEarly warning signals in the adaptive immune system originate from T Cell recognition of pathogenic antigens on the surface of Antigen Presenting Cells (APCs). T Cell activation and the production of warning molecules (cytokines) proceeds downstream of antigen binding to T Cell Receptor (TCR) at T Cell:APC membrane interfaces. Complex proofreading mechanisms are required to control T Cell activation, and this process is thought to take place primarily via TCR proximal signaling events on the inner T Cell membrane. A number of critical components for the origination of T Cell activation signals are well known, including the membrane-associated scaffold LAT that becomes phosphorylated following TCR-antigen binding events. LAT recruits a web of cytosolic adapters, like Grb2, and signaling enzymes, such as Sos1 and PLCγ1. LAT has been recognized as a signaling hub wherefrom both proliferation signals, through Sos1 activation of Ras, and cytokine production signals, through PLCγ1-mediated Calcium flux, diverge. Additionally, this hub has been established as a biomolecular membrane protein condensate, with multivalent crosslinking through Grb2 and Sos1 interactions as well as more recently discovered crosslinking through Grb2 dimerization and the SH array of PLCγ1. The consequences of condensation are not yet fully understood, but in new accounts from our lab, it has been suggested to encode information relevant for T Cell activation. Vital to the study of this system, we have established an in vitro platform for the study of fluorescently labeled proteins on supported bilayers with visualization through TIRF microscopy. LAT functionalized to these bilayers is capable of both recruitment of solution proteins and forming condensates, and signals from recruited enzymes can be detected using labeled biosensors. This allows for the piece-wise investigation of mechanisms in the formation of this central hub. Herein, I cover our recent efforts to increase reproducibility in the use of this platform and expound on the recent developments in the modes of LAT crosslinking leading to condensation. Then, I demonstrate the effect of LAT condensation on active PLCγ1 from mammalian cell lysate, most notably a marked decrease in activity in pLAT:Grb2 condensates. I conclude with single particle tracking experiments of two Ras isoforms that reveal functional differences in their interactions with the membrane as a function of lipid composition. These stand to be important contributions to the understanding of TCR signaling and will empower future discovery
Recommended from our members
Spatiotemporal signaling dynamics evoked by single binding interactions between a peptide-bound MHC molecule and a T cell receptor
Full text linkT cells (TCs) have the crucial immunological task of discriminating foreign peptide fragments from endogenous (self) peptide fragments. This recognition is carried out by the T Cell Receptor (TCR) on the TC surface, which binds to an antigen-derived peptide fragment presented by major histocompatibility complex (pMHC) on the surface of an Antigen Presenting Cells (APC). Despite the centrality of this TCR:pMHC interaction and the myriad techniques used analyze it, few studies exist in which the signaling consequences of individual TCR:pMHC binding events have been determined. Direct examination of the outcomes from single TCR:pMHC encounters provides insight into how the stochastic signaling network of the T cell performs crucial tasks such as signal integration and ligand discrimination. In Chapter 1, individual TCR:pMHC binding interactions (dwell times) are mapped to two proximal events in T cell activation: the assembly of Linker for Activation of T cells (LAT) molecules; and, translocation of Nuclear Factor of Activated T cells (NFAT). After TCR engagement with agonist pMHC, the LAT scaffold protein forms a hub of signaling from which a number of subsequent signaling steps are initiated; hence, LAT assembly is a gatekeeper of downstream signaling. I observe that the probability of LAT assembly formation, localized to ligated TCR, is an increasing function of the dwell time of the TCR:pMHC interaction, suggesting that the formation of LAT assembly from ligated TCR represents a form of kinetic proofreading; however, the size and lifetime of a LAT assembly is independent of the TCR:pMHC dwell time interaction that produced it. This discreteness of LAT clustering from TCR:pMHC dwell time has consequences for signal integration from multiple binding events. In Chapter 2, I explore what distinguishes a productive TCR:pMHC binding event from one that fails to trigger LAT assembly, as an approach to delineate the “kinetic bottlenecks” in antigen discrimination and T cell activation. I found that activation of zeta-chain- associated protein kinase of 70 kDa (ZAP-70), which phosphorylates LAT at multiple sites, is necessary, but insufficient. Aside from ZAP-70 activity, diffusive phospho-LAT requires an additional event to trigger aggregation. Importantly, I found that, at low pMHC densities, PLC-γ1 controls the timing for the initiation of LAT clustering, a dependency not previously identified. One feature I readily observed in my experiments is that LAT assemblies can form without any apparent originating pMHC. The composition and function of these apparent orphan LAT assemblies are investigated preliminarily in Chapter 3. In summary, by measuring the correlation between single receptor (TCR)-ligand (pMHC) binding events and subsequent steps in T cell activation, one obtains unprecedented quantitative insight into the stochastic nature of signal transduction in T cells
Recommended from our members
Spatio-mechanical EphA2/ephrin-A1 Signaling in Cancer Cells
Full text linkCommunication strategies in nature are an integral part to the survival of multi-cellular organisms. Cell membranes provide the chemical environment in which intercellular signaling begins. The vast complexity of this signaling requires that a relatively conserved set of chemical constituents be able to generate enormous signal diversity. Spatial sorting of signaling molecules within the membrane allows for this diversity. My research uses synthetic lipid membranes, solid-state nanostructures, and high-resolution imaging to study a potentially novel spatio-mechanical regulatory mechanism in the EphA2 signaling pathway. My hypothesis is that the multi-scale organization of the EphA2 receptor in the cell membrane regulates its biochemical function. This hypothesis is motivated by the idea that extracellular mechanical inputs have an important role in intracellular signaling cascades
Recommended from our members
Study of signal transduction in reconstituted membranes: Ras activation by SOS and RasGRP1
Full text linkCellular membranes organize signal transduction, serving as platforms for protein interactions, as well as direct modulators of enzyme function. Since many signaling reactions converge at the plasma membrane, understanding the coupling between proteinfunction and membrane localization of signaling complex is of fundamental importance. However, a quantitative description of many of such interactions has been lacking, largely due to the difficulty in performing measurements in the complex cellular environment. Here, we reconstitute and quantitatively probe the kinetics between small GTPase Ras and its activators: Son of Sevenless (SOS) and Ras guanyl nucleotide-releasing protein(RasGRP) using supported membranes.The activation of the membrane-associated Ras by cytosolic guanine nucleotide exchange factors, SOS and RasGRP, is a key step in a plethora of receptor-mediated cell signaling pathways. Both SOS and RasGRP contain multiple domains and several of whichare known to regulate the activation of SOS/RasGRP. In this dissertation, we study in parallel the kinetics of SOS and RasGRP-catalyzed Ras activation as well as the mechanisms that release the autoinhibition of SOS/RasGRP at single molecule level.We develop an assay with supported bilayers functionalized with H-Ras to monitor the enzymatic activities from hundreds of individual SOS proteins. The data reveals that SOS is dynamically heterogeneous, sampling a broad distribution of turnover rates viastochastic fluctuations between distinct states. The regulatory domains allosterically inhibit SOS by suppressing fluctuations to high activity states with sensitivity to both Ras nucleotide state and lipids. On the subject of Ras activation by RasGRP, the functional role of lipid interacting C1 domain of RasGRP1 has been studied. The preliminary results suggest that diacylglycerol (DAG), through the interaction with the C1 domain, regulates the function of RasGRP1 primarily by increasing its membrane localization
- …
