Browsing by Subject "FRET"

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects over 5 million people in the United States alone. This number is predicted to triple to by the year 2050 due to both increasing life expectancies and the absence of disease-attenuating drugs. The etiology of AD remains unclear, and although there are multiple theories implicating everything from oxidative stress to protein misfolding, misregulated metal ions appear as a common thread in disease pathology.

Chelation therapy has shown some effectiveness in clinical trials, but to date, there are no FDA-approved metal chelators for the treatment of AD. One of the biggest problems with general chelators is their inability to differentiate between the metal ions involved in disease progression verses those involved in normal metabolic function. To address this problem, we have developed a prochelator approach whereby the prochelator (SWH) does not bind metals with significant biological affinity. However, once activated to the chelator (CP) via enzymatic hydrolysis, the molecule is able to bind copper and reduce its toxicity both in vitro and in a cellular model of Alzheimer's Disease.

Central to this strategy is the site-specificity provided by enzymatic activation of the prochelator. In our system, SWH to CP conversion is mediated by beta-secretase, an enzyme involved in A-beta generation. However, in order to render SWH capable of hydrolysis in cells, we modified the prochelator to contain a dihydrocholesterol membrane anchor attached via a polyethylene glycol linker. From this construct, we created beta-MAP, which is an SWH-based FRET probe to demonstrate beta-secretase-mediated conversion of SWH to CP. beta-MAP was also used to confirm the efficacy of a known beta-secretase inhibitor without the need to for mutated cells lines or expensive antibodies. beta;-MAP and the associated microscopy method represent a significant advancement to the currently available ELISA assays for beta-secretase activity.

While activation of the prochelator by an enzyme in cells is encouraging, non-specific hydrolysis of the peptide prevents significant accumulation of the chelator on the cell membrane. Furthermore, attachment of the polyethylene glycol and sterol units induce cell toxicity not seen with the native CP peptide. These drawbacks prevent the current prochelator from effectively protecting cells from AD conditions. Structural modifications to overcome these problems, including implementation of a new peptide sequence are planned for future experiments.

Mechanical forces are potent drivers of many biological processes. The form and function of many tissues depends on cells receiving the proper mechanical signals either from neighboring cells or from the underlying matrix. During development, dynamic tissue movements are driven by cell contractility and stem cell fate depends in large part to the mechanical forces they feel in their local surroundings. Conversely, aberrant mechanosensitive signaling is associated with the pathological progression of several disease states, such as cancer and atherosclerosis, for which effective treatments are scarce. As such, understanding how cells physically interact with and detect mechanical aspects of their microenvironment is critical to both understanding developmental processes and developing new treatments for disease.

Mechanical information from the microenvironment is converted into biochemical signals inside cells through molecular scale processes, collectively referred to as mechanotransduction. Many of the events associated with mechanosensitive signaling and mechanotransduction are mediated by force-dependent changes in protein structure and function. However, the lack of available tools to study these molecular scale processes in cells is currently preventing further progress. To address this need, the goals of this dissertation were to (1) improve upon and expand the capabilities of existing tools to visualize molecular forces and (2) develop novel methodologies to detect force-sensitive signaling events inside cells.

We began by focusing on the further development and improvement of one of the most critical tools to mechanobiological investigations: Förster Resonance Energy Transfer (FRET)-based molecular tension sensors. While these sensors have contributed greatly to our understanding of mechanobiology, the limited dynamic range and inability to specify the mechanical sensitivity of existing sensors has hindered their use in diverse cellular contexts. Through both experiments and modeling efforts, we developed a comprehensive biophysical understanding of molecular tension sensor function that enabled the creation of new sensors with predictable and tunable mechanical sensitivities. We used this knowledge to create a sensor optimized to study the ~1-6pN loads experienced by vinculin, a critical linker protein that plays an integral role in connecting cells, via focal adhesions (FAs), to the extracellular matrix (ECM). Using this optimized sensor enabled sensitive detection of changes in molecular loads across single cells and even within individual FA structures. We also expanded the capabilities of tension sensors to investigate the potentially distinct roles of protein force and protein extension in activating mechanosensitive signaling. Specifically, a trio of these new biosensors with distinct force- and extension-sensitivities revealed that an extension-based control paradigm underlies cellular control of vinculin loading.

Since these sensors uniquely provide insight into which molecules are physically engaged and could be participating in mechanically-based signaling, we chose to investigate which cytoskeletal structures mediate patterns of vinculin loading at multiple length scales within the cell. Specifically, we focused on two active, but distinct force generating machineries inside cells: stress fibers (SFs) and lamellipodial protrusions (LPs). By measuring vinculin tension in various mechanical and biochemical contexts, we found significant evidence for vinculin’s involvement in force transmission from both LP and SF structures. However, the distribution of loads across vinculin at the level of a single FA was dramatically different between these two distinct actin structures. Specifically, asymmetric distribution of vinculin load along individual FAs was an exclusive feature of SF-associated FAs. Subsequent experiments showed that formation and maintenance of these gradient loading profiles also depends on vinculin’s interactions with key binding partners, suggesting that both the magnitude as well as the pattern of vinculin loading within FAs are independently regulated by cells, and thus might serve distinct purposes in cellular mechanosensing.

Towards understanding the biochemical consequences of protein load at the molecular level, we developed an imaging-based technique to detect one of the events most often implicated in mechanically-based signal transduction: the formation of force-sensitive protein-protein interactions (PPIs). While these force sensitive interactions have been extensively documented in vitro, the extent to which they occur inside living cells is debated. This imaging-based technique, which we refer to as fluorescence-force co-localization (FFC), involves simultaneous FRET imaging of a FRET-based tension sensor to visualize protein loading and correlate this with the recruitment of other species to areas of high molecular loads. With vinculin as a prototypical example and a screen-based approach in mind, we used immunofluorescence to measure the relative enrichment of 20 other key FA proteins in areas of high vinculin tension. Factoring in what we previously learned about (1) the importance of actin architecture and (2) the well-established role of vinculin alone in controlling FA composition, we provide a multiparametric perspective on a potential mechanotransduction node associated with high vinculin loads. Focusing on the top five hits from this FFC screen, subsequent experiments revealed a genuine vinculin tension-dependent interaction with migfilin. While the involvement of both vinculin and migfilin in cardiac settings is a tempting line of future work, the work presented in this dissertation even more powerfully provides a proof-of-principle for the detection of force-sensitive PPIs in cells.

In total, the techniques developed in this dissertation enable detection of multiple molecular events associated with mechanotransduction inside cells. The improvement of FRET-based tension sensors as well as the ability to define their mechanical properties a priori should expedite investigations of molecular forces in diverse biological contexts. Additionally, the realization of force-dependent PPIs inside cells provided by the FFC screen constitutes a significant step towards uncovering mechanically-based signaling mechanisms inside cells. The more widespread application of these tools will undoubtedly fuel our understanding of mechanotransduction and could enable better control of cell behaviors in engineered tissues as well as the development of treatments for mechanosensitive diseases.

The coordinated movement of groups of cells, called collective cell migration (CCM), plays important roles in many developmental, physiological, and pathological processes. During CCM, cells remain mechanically coupled to their neighbors, which enables both long-range coordination and local rearrangements. This coupling requires the ability of cell adhesions to transmit and adapt to mechanical forces. However, the molecular mechanisms that underly these mechanosensitive processes remain poorly understood, hindering efforts to manipulate CCM for therapeutic or engineering purposes. To address this gap, this dissertation develops and applies a combination of experimental and modeling approaches to investigate molecular scale mechanosensitive processes. In the first part of this dissertation, we asked how mechanical forces and biochemical regulation interact to control mechanical coupling during CCM. We focused on the mechanical linker protein vinculin, which is known to mediate adhesion strengthening. Using a set of Förster resonance energy transfer (FRET)-based biosensors, we probed the mechanical function and biochemical regulation of vinculin, elucidating a switch that toggles both the activation and molecular loading of vinculin at cell adhesions. We found that the vinculin switch controlled both the speed and coordination of CCM, resulting in a covariation of these variables that suggested changes in adhesion-based friction. To bridge molecular and cellular measurements, we developed molecularly specific models of frictional forces at cell adhesions based on the force-sensitive bond dynamics of key proteins. In these models, increases in vinculin activation and loading produced increases in friction at adhesion structures, and this was due to the engagement of vinculin-actin catch bonding. Together, this work reveals how the biochemical regulation of a linker protein (vinculin) affects a cell-level mechanical property (adhesion-based friction) to control a multicellular behavior (CCM).

In the second part of this dissertation, we focused on how cells sense mechanical forces at the molecular scale. This is thought to occur by force-induced changes in the structure/function of proteins. However, how forces affect protein function inside cells remains poorly understood due to a lack of tools to probe this inside cells. Motivated by in vitro work showing that the mechanical loading of fluorescent proteins (FPs) causes a reversible switching of their fluorescence, we investigated if this phenomenon could be detected inside cells to directly visualize force-sensitive protein function. Using a mathematical model of FP mechanical switching, we developed a framework to detect it inside FRET-based biosensors. Applying this framework, we observed FP mechanical switching in two sensors, a synthetic actin-crosslinker and the linker protein vinculin, and we found that mechanical switching was altered by manipulations to cellular forces on the sensor as well as force-dependent bond dynamics of the sensor. Together, this work develops a new framework for assessing the mechanical stability of FPs and enables visualizing the effect of forces on protein function inside cells.

Overall, the work in this dissertation advances our basic understanding of mechanosensitive processes, addressing knowledge gaps in CCM and mechanobiology. The frameworks we have developed for integrating molecular- and cellular-level experiments with mathematical models will facilitate new mechanistic studies into mechanosensitive processes involving other proteins and biological contexts.

FRET between pairs of fluorophores is widely used as a biological assay. However, the properties of larger fluorophore networks are poorly understood and their application space has not yet been fully explored. This dissertation introduces DNA self-assembled FÖrster Resonance Energy Transfer (FRET) networks that provide a unique optical output when probed by a series of light pulses. We create a Markov model of the FRET networks and analyze over 1200 time-resolved fluorescence measurements on 300 prototypical networks. Our results show that the optical responses of FRET networks are highly repeatable and minor variations between the FRET networks can be discriminated resulting in a total of 10375 unique responses. These results are used in the following breakthrough applications:

1. Unclonable Cryptographic Key for Secure Authentication and Communication

Modern authentication protocols rely on an asymmetry in the effort required by a legitimate user and an adversary to accurately decrypt an encoded message. These protocols ensure that communication between legitimate users is possible in polynomial time using a private key but a user without access to the exact key cannot compute the function using a probabilistic polynomial-time algorithm. Private-key cryptographic techniques currently employ physical keys based on algorithmic one-way functions, which are conjectured mathematical objects that are easy to compute but difficult to invert. Well-known examples of such one-way functions include the RSA and the Rabin functions. Although algorithmic one-way functions are widely used for authentication, their reliance on computational difficulty to provide security implies that they are not protected against future advances in computational capacity or speed. Also, use of a highly parallel network of conventional computers could potentially reverse engineer a key from the challenge-response pairs used in past communications. The key may also be obtained by duplicating the device. Most of the current physical embodiments of algorithmic one-way functions come with a tamper resistant packaging but remain vulnerable to sophisticated attacks.

We develop a RET based physical key to overcome the limitations of conventional security keys. The key exploits resonance energy transfer between a network of fluorophores placed on a nanostructure. The fluorophores provide a unique, unpredictable output when probed by a series of light pulses of specific wavelengths and delays. A critical advantage of the RET key over existing keys is that the manufacturing process allows two identical devices to be produced allowing us to exploit the advantages of symmetric key encryption, for the first time, without the need to physically transfer the device between the two communicating parties.

It is infeasible to model, characterize or replicate our key using modern cryptographic attacks including unfettered physical access to the device. This is because of the difficulty in characterizing the nanoscale structure and the large number of challenge-response pairs achievable for each key. Atomic force microscopy and time -resolved fluorescence measurements are performed to characterize the nanoscale structures. From over 1200 measurements on 300 prototypical keys, we estimate that a legitimate user would have a computational advantage of 10340 years over an attacker even if the attacker uses all the computational resources available in the world. Thus, the computational advantage of our key ensures perfect theoretical security for the foreseeable future. We provide an authentication protocol for use of the key and demonstrate that legitimate users are successfully authenticated 99.48% of the time with two trials.

2. Multiplexed Fluorescence Sensor for Cancer Detection

Fluorescence microscopy is one of the most widely used assays in biological systems. However, the technique suffers from limited multiplexing capability with previous attempts at detecting more than 11 fluorophores simultaneously resulting in barcodes that are too big for in vivo analysis, expensive and involve time-consuming detection schemes. Here, we introduce DNA self-assembled FRET networks that provide a unique, optical output when probed by a series of light pulses. Markov and entropy modeling of the nanoscale FRET sensors show that 125 fluorophores can be observed simultaneously. Furthermore, experimental analyses of over 1200 time-resolved fluorescence signatures show that the optical responses are repeatable 99.48% of the time and that minor variations between FRET networks can be discriminated resulting in a total of 10375 unique responses. This enormous increase in spatial information density enabled by FRET networks allowed us to identify molecular signatures in lung and breast cancer tumors.

It is now known that the presence of aberrant DNA/RNA secondary structure in the regulatory regions of genes involved in cell proliferation, cells growth and apoptosis can lead to cancer. The FRET sensor we designed, self-assembles DNA probes labeled with acceptor fluorophores to the target DNA/RNA secondary structure forming an optical network. A DNA strand labeled with a donor fluorophore triplex binds to a unique sequence adjacent to the secondary structure. When the donor fluorophore is excited, the optical network results in a different optical signal based on the presence of the wild-type or the aberrant secondary structure, through which we identified lung and breast cancer cells with high specificity and over 99.9% repeatability. The small size of fluorophores results in molecular scale spatial resolution while the optical sensing mechanism enables in vitro and in vivo characterization of the structure at picosecond resolution.

Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive.

Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity.

We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.

Cell migration and multicellular interactions are essential for the formation and maintenance of tissue structure. The dysregulation of these processes also contributes to developmental defects and pathological processes. A prominent question is how biochemical and biophysical information, which acts at the level of an individual cell, is transmitted and integrated by neighboring cells to yield coordinated behavior. In a process known as collective cell migration (CCM), mechanical coupling of cells is thought to play a key role in coordinating migration across many cell lengths. Mechanocoupling refers to the mechanical integration of cell-cell adhesions and the contractile actomyosin network. While pertinent signaling pathways have been identified that mediate CCM, the mechanisms involved in mechanocoupling at the molecular level are poorly understood. Progress in the field has been limited due to the molecular complexity of adhesion structures and technical limitations of measuring in vivo mechanics to identify mechanosensitive elements. Therefore, a central but understudied phenomenon in cell migration is the study of mechanocoupling. The overall premise of this proposal is that we can use a new type of force-sensitive biosensor to identify proteins responsible for mediating mechanocoupling. The advances from this approach will fundamentally advance our understanding of CCM and open new doors for the manipulation and control of CCM.

The force-sensitive biosensor used in this work was a Fӧrster resonance energy transfer (FRET)-based tension sensor, which enables the measurement of molecular-scale forces across proteins based on changes in emitted light. We focused specifically on the role of vinculin in mediating mechanocoupling for two important reasons. Firstly, vinculin is the only protein known to localize to both FAs and AJs in response to mechanical loading. Secondly, vinculin activity can be regulated by multiple kinases through site-specific phosphorylation. However, the implications of vinculin regulation by these kinases has not been fully elucidated. As the reliability and reproducibility of measurements made with FRET-based tension sensors has not been thoroughly examined, we first developed numerical methods that improve the accuracy of measurements made using sensitized emission-based imaging. To establish that FRET-based tension sensors are versatile tools that provide consistent measurements, we then used these methods to demonstrate that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggested FRET-based tension sensors could be coupled with a variety of immuno-fluorescent labeling techniques for future investigations into mechanocoupling. Additionally, as tension sensors are frequently employed in complex biological samples where large experimental repeats may be challenging, we examined how sample size affects the uncertainty of FRET measurements. In total, this groundwork established useful guidelines to ensure precise and reproducible measurements for studying mechanics in CCM using FRET-based tension sensors.

To investigate the mediators of mechanocoupling in CCM, epithelial sheet migration was studied because it is characterized by long-range coordination and, presumably, high mechanocoupling. Two epithelial cell lines were subjected to a non-wounding 2D migration assay and found to exhibit stark differences in migratory characteristics, including speed and velocity correlations. The pertinent subcellular structures for mechanocoupling, namely focal adhesions (FAs), adherens junctions (AJs), and the actomyosin cytoskeleton, appeared to contribute to these differences. A significant finding was that actin belts, traditionally associated with long-range coupling in developmental events, did not lead to global coordination within a migrating layer. Instead, measurements of vinculin tension demonstrated that vinculin mechanocoupling was associated with long-range coordination throughout a migrating layer and the formation of a pluricellular actin network. Interestingly, vinculin was shown to act as a mechanocoupler throughout a cell’s cytoplasmic actin network, demonstrating a previously unappreciated role of vinculin. Universally, vinculin mechanocoupling involved actin interactions and required a head-specific site known to interact with a variety of binding partners including talin, β-catenin, α-catenin, and α-actinin. As vinculin can undergo head-tail autoinhibition, its conformation was evaluated. These findings indicated that vinculin was differentially regulated. By probing the role of three kinases, it was found that serine phosphorylation by Protein Kinase C (PKC) is an important regulator of vinculin mechanocoupling.

In summary, we propose that long-range coordination during CCM can be mediated by mechanocoupling of a supracellular actin network. Based on our findings, vinculin mechanocoupling is associated with the emergence of this supracellular network. Furthermore, serine phosphorylation appears to play a previously underappreciated role in regulating the mechanical integration of migrating cells. These advancements serve as an important step toward better understanding the physical mechanisms of CCM.