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- All Subjects: Biochemistry
- Creators: Borges, Chad
- Creators: School of Life Sciences
Description
Photosystem I (PSI) is a multi-subunit, pigment-protein complex that catalyzes light-driven electron transfer (ET) in its bi-branched reaction center (RC). Recently it was suggested that the initial charge separation (CS) event can take place independently within each ec2/ec3 chlorophyll pair. In order to improve our understanding of this phenomenon, we have generated new mutations in the PsaA and PsaB subunits near the electron transfer cofactor 2 (ec2 chlorophyll). PsaA-Asn604 accepts a hydrogen bond from the water molecule that is the axial ligand of ec2B and the case is similar for PsaB-Asn591 and ec2A. The second set of targeted sites was PsaA-Ala684 and PsaB-Ala664, whose methyl groups are present near ec2A and ec2B, respectively. We generated a number of mutants by targeting the selected protein residues. These mutations were expected to alter the energetics of the primary charge separation event.
The PsaA-A684N mutants exhibited increased ET on the B-branch as compared to the A-branch in both in vivo and in vitro conditions. The transient electron paramagnetic resonance (EPR) spectroscopy revealed the formation of increased B-side radical pair (RP) at ambient and cryogenic temperatures. The ultrafast transient absorption spectroscopy and fluorescence decay measurement of the PsaA-A684N and PsaB-A664N showed a slight deceleration of energy trapping. Thus making mutations near ec2 on each branch resulted into modulation of the charge separation process. In the second set of mutants, where ec2 cofactor was target by substitution of PsaA-Asn604 or PsaB-Asn591 to other amino acids, a drop in energy trapping was observed. The quantum yield of CS decreases in Asn to Leu and His mutants on the respective branch. The P700 triplet state was not observed at room and cryogenic temperature for these mutants, nor was a rapid decay of P700+ in the nanosecond timescale, indicating that the mutations do not cause a blockage of electron transfer from the ec3 Chl. Time-resolved fluorescence results showed a decrease in the lifetime of the energy trapping. We interpret this decrease in lifetime as a new channel of excitation energy decay, in which the untrapped energy dissipates as heat through a fast internal conversion process. Thus, a variety of spectroscopic measurements of PSI with point mutations near the ec2 cofactor further support that the ec2 cofactor is involved in energy trapping process.
The PsaA-A684N mutants exhibited increased ET on the B-branch as compared to the A-branch in both in vivo and in vitro conditions. The transient electron paramagnetic resonance (EPR) spectroscopy revealed the formation of increased B-side radical pair (RP) at ambient and cryogenic temperatures. The ultrafast transient absorption spectroscopy and fluorescence decay measurement of the PsaA-A684N and PsaB-A664N showed a slight deceleration of energy trapping. Thus making mutations near ec2 on each branch resulted into modulation of the charge separation process. In the second set of mutants, where ec2 cofactor was target by substitution of PsaA-Asn604 or PsaB-Asn591 to other amino acids, a drop in energy trapping was observed. The quantum yield of CS decreases in Asn to Leu and His mutants on the respective branch. The P700 triplet state was not observed at room and cryogenic temperature for these mutants, nor was a rapid decay of P700+ in the nanosecond timescale, indicating that the mutations do not cause a blockage of electron transfer from the ec3 Chl. Time-resolved fluorescence results showed a decrease in the lifetime of the energy trapping. We interpret this decrease in lifetime as a new channel of excitation energy decay, in which the untrapped energy dissipates as heat through a fast internal conversion process. Thus, a variety of spectroscopic measurements of PSI with point mutations near the ec2 cofactor further support that the ec2 cofactor is involved in energy trapping process.
ContributorsBadshah, Syed Lal (Author) / Redding, Kevin E (Thesis advisor) / Fromme, Petra (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014
Description
The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore, I focus on the evolution of Kaede-type FPs from Faviina suborder ancestral FP. A total of 13 mutations have been identified previously that recapitulate the evolution of Kaede-type red FPs from the ancestral green FP. To examine the effect of each mutation, total ten reconstructed FPs were analyzed and six x-ray crystal structures were solved. These substitutions created a more hydrophilic environment around the carbonyl group of Phe61. Also, they increased the flexibility of the c-terminal chain, which keeps it from interacting with the entrance of the putative solvent channel. The photoconversion reaction shows a twophase kinetics. After the rapid initial phase, the overall reaction followed the firstorder kinetics. Based on the crystal structure analysis, I propose a new mechanism for Kaede-type FP photoconversion process, which a proton transfers via Gln38 to the carbonyl group of Phe61.
ContributorsKim, Hanseong (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Redding, Kevin E (Committee member) / Arizona State University (Publisher)
Created2012
Description
Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational self-modification. Its value during chromophore maturation has been demonstrated by mutational studies and a hypothesis proposed for its catalytic function. A strategy is described herein to determine its pKa value via NMR to determine whether Arg96 possesses the chemical capacity to function as a general base during GFP chromophore biosynthesis. Förster resonance energy transfer (FRET) techniques commonly employ Enhanced Cyan Fluorescent Proteins (ECFPs) and their derivatives as donor fluorophores useful in real-time, live-cell imaging. These proteins have a tryptophan-derived chromophore that emits light in the blue region of the visible spectrum. Most ECFPs suffer from fluorescence instability, which, coupled with their low quantum yield, makes data analysis unreliable. The structural heterogeneity of these proteins also results in undesirable photophysical characteristics. Recently, mCerulean3, a ten amino acid mutant of ECFP, was introduced as an optimized FRET-donor protein (1). The amino acids changed include a mobile residue, Asp148, which has been mutated to a glycine in the new construct, and Thr65 near the chromophore has been mutated to a serine, the wild-type residue at this location. I have solved the x-ray crystal structure of mCerulean3 at low pH and find that the pH-dependent isomerization has been eliminated. The chromophore is in the trans-conformation previously observed in Cerulean at pH 8. The mutations that increase the quantum yield and improve fluorescence brightness result in a stable, bright donor fluorophore well-suited for use in quantitative microscopic imaging.
ContributorsWatkins, Jennifer L (Author) / Wachter, Rebekka M. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2012
Description
Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery.
ContributorsRai, Samita (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Borges, Chad (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type is altered upon environmental stimulations, influencing the bacterial survival and virulence within the host tissue. Therefore, it is important to explore methods to identify the sub-populations of Salmonella.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
This work exploits insulator-based dielectrophoresis (iDEP) for the manipulation of mitochondria and Salmonella. The iDEP migration and trapping of mitochondria were investigated under both DC and low-frequency AC conditions, establishing that mitochondria exhibit negative DEP. Also, the first realization of size-based iDEP sorting experiments of mitochondria were demonstrated. As for Salmonella, the preliminary study revealed positive DEP behavior. Distinct trapping potential thresholds were found for the sub-populations with different surface charges.
Further, DEP was integrated with a non-intuitive migration mechanism termed absolute negative mobility (ANM), inducing a deterministic trapping component which allows the directed transport of µm- and sub-µm sized (bio)particles in microfluidic devices with a nonlinear post array under the periodic action of electrokinetic and dielectrophoretic forces. Regimes were revealed both numerically and experimentally in which larger particles migrate against the average applied force, whereas smaller particles show normal response. Moreover, this deterministic ANM (dANM) was characterized with polystyrene beads demonstrating improved migration speed at least two orders of magnitude higher compared to previous ANM systems with similar sized colloids. In addition, dANM was induced for mitochondria with an AC-overlaid waveform representing the first demonstration of ANM migration with biological species. Thus, it is envisioned that the efficient size selectivity of this novel migration mechanism can be employed in nanotechnology, organelle sub-population studies or fractionating protein nanocrystals.
ContributorsLuo, Jinghui (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.
In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
ContributorsGisriel, Christopher J (Author) / Redding, Kevin E (Thesis advisor) / Jones, Anne K (Committee member) / Allen, James P. (Committee member) / Arizona State University (Publisher)
Created2017
Description
Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity.
ContributorsSerban, Andrew J (Author) / Wachter, Rebekka M. (Thesis advisor) / Levitus, Marcia (Thesis advisor) / Redding, Kevin E (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2018
Description
To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given that menaquinone is active within the heliobacterial reaction center in some way. Purified heliobacterial reaction center was reconstituted into the liposomes and a recombinant cytochrome c553 was decorated onto the liposome surface. The native lipid-attachment sequence of cytochrome c553 was truncated and replaced with a hexahistidine tag. Thus, the membrane-anchoring observed in vivo was simulated through the histidine tag of the recombinant cytochrome binding to the Ni(II)-NTA lipid's head group. The kinetics of electron transfer in this system was measured and compared to native membranes using transient absorption spectroscopy. The preferential-orientation of reconstituted heliobacterial reaction center was also measured by monitoring the proteoliposome system's ability to reduce a soluble acceptor, flavodoxin, in both whole and detergent-solubilized proteoliposome conditions. These data demonstrate that this proteoliposome system is reliable, biomimetic, and efficient for selectively testing the function of the photosynthetic reaction center of Heliobacterium modesticaldum and its interactions with both donors and acceptors. The recombinant cytochrome c553 performs similarly to native cytochrome c553 in heliobacterial membranes. These data also support the hypothesis that the orientation of the reconstituted reaction center is inherently selective for its bacteriochlorophyll special pair directed to the outer-leaflet of the liposome.
ContributorsJohnson, William Alexander (Author) / Redding, Kevin E (Thesis advisor) / Van Horn, Wade D (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological systems have long been known to utilize two processes for energy conservation: substrate-level phosphorylation and electron transport phosphorylation. Recently, a new bioenergetic process was discovered that increases ATP yields: flavin-based electron bifurcation (FBEB). This process couples an energetically favorable reaction with an energetically unfavorable one to conserve energy in the organism. Currently, the mechanisms of enzymes that perform FBEB are unknown. In this work, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn), a FBEB enzyme, is used as a model system to study this phenomenon. Nfn is a heterodimeric enzyme that reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with the endergonic reduction of NADP+ by NADH. Protein film electrochemistry (PFE) has been utilized to characterize the catalytic properties of three ferredoxins, possible substrates for Nfn enzymes, from organisms that perform FBEB: Pyrococcus furiosus (PfFd), Thermotoga maritima (TmFd), and Caldicellulosiruptor bescii (CbFd). Additionally, PFE is utilized to characterize three Nfn enzymes from two different archaea in the family Thermococcaceae: two from P. furiosus (PfNfnI and PfXfn), and one from Thermococcus sibiricus (TsNfnABC). Key results are as follows. The reduction potentials of the [4Fe4S]2+/1+ couple for all three ferredoxins are pH independent and modestly temperature dependent, and the Marcus reorganization energies of PfFd and TmFd are relatively small, suggesting optimized electron transfer. Electrocatalytic experiments show that PfNfnI is tuned for NADP+ reduction by both fast rates and a low binding constant for NADP+. A PfNfnI variant engineered to have only cysteines as coordinating ligands for its [FeS] clusters has significantly altered rates of electrocatalysis, substrate binding, and FBEB activity. This suggests that the heteroligands in the primary coordination sphere of the [FeS] clusters play a role in controlling catalysis by Nfn. Furthermore, a variant of PfNfnI lacking its small subunit, designed to probe allosteric effects at the bifurcating site, has altered substrate binding at the NADP(H) binding site, i.e. the bifurcation site. PfXfn and TsNfnABC, representing different types of Nfn enzymes, have different electrocatalytic properties than PfNfnI, including slower rates of FBEB. This suggests that Nfn enzymes vary significantly over phylogenetically similar organisms despite relatively high primary sequence homology.
ContributorsJennings, David Peter (Author) / Jones, Anne K (Thesis advisor) / Redding, Kevin E (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2018
Description
Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined.
Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model.
Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces.
ContributorsZhang, Fenni (Author) / Tao, Nongjian (Thesis advisor) / Chae, Junseok (Committee member) / Borges, Chad (Committee member) / Jing, Tianwei (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018