An important part of addiction recovery is degrading high value associations between drug cues and the drugs themselves. Dopamine plays a crucial role in learning, and is specifically implicated in the prefrontal cortex (PFC) and reversal learning - learning to update and change behavior when it is no longer being rewarded. Past studies have reported elevations in dopamine during contingency reversal, but the timescale of how activity of PFC dopamine neurons maps to reversal learning remains unclear. Here we investigated the activity of PFC dopamine during reversal learning in a longitudinal fiber photometry study, recording dopamine signal on a timescale of seconds. Mice were trained on a reversal learning task where they initially learned that two of four presented odors precipitated a sucrose reward in 85% of the trials while the remaining two odors precipitated the reward for only 15% of the trials. Once the learning was stable, reward probability flipped for two odors (one 85% odor and one 15% odor) and the mice had to update their behavior to the new odor/reward structure. Fiber photometry recordings were conducted during pre-reversal, reversal, and post-reversal stages of the study. Our data replicate findings demonstrating elevated dopamine release during the reversal period, centered around the 15-85 cue. Analysis of the relationship between the dopamine signal and behavior also revealed significant cue, reward prediction error, and 15-85 reversal coding in the majority of animals, suggesting a multi-faceted role for dopamine in the PFC. Given this, dopamine in the PFC may play an important mediating role in the enhancement of associations between drugs and drug cues, but does not play a clear role in contingency degradation.

The use of opioid drugs for pain management in postoperative settings has been widespread since the 1860s. However, the risk factor for developing an opioid use disorder (OUD) has increased substantially with its continued use, with addiction rates of more than 10% for those taking opioids. An obstacle to abstinence in opioid addiction are the adverse side effects that occur during cessation of drug use once dependence has formed, including nausea, anxiety, vomiting, and depression. The Nucleus Accumbens (NAc) is part of the mesocorticolimbic reward pathway. Decades of pharmacological studies demonstrate that nearly all abused drugs evoke dopamine release within the NAc, thus altering innate systems for how reward is processed. The activity of NAc neurons is strongly regulated by efferent excitatory input from numerous brain regions. The paraventricular thalamus (PVT) a relatively understudied brain region, regulates behavioral responses to reward and aversive stimuli as well as to drugs of abuse such as morphine. Our preliminary data demonstrate that the activity of these projections is highly regulated by the Cannabinoid 1 Receptor (CB1), which mediates the primary psychoactive effect of cannabis. This is particularly relevant on account of recent clinical findings demonstrating that activation of CB1 can ameliorate the aversive effects of opiate withdrawal. Using fiber photometry (which uses fluorescence emission of the calcium sensitive fluorophore, GCaMP, as a proxy measurement for neural activity), I have shown that this circuit is activated by aversive stimuli and inhibited by rewarding stimuli. Furthermore, treatment with morphine can attenuate the pain-induced activation of this circuit. However, whether cannabinoids can influence this circuit's activity to reduce withdrawal symptoms remains untested. Our research will contribute to our understanding of the neurophysiological basis for opiate withdrawal and how cannabinoids could represent a novel class of therapeutics for the treatment of opiate use disorder.

Addiction is characterized by the compulsive use of substances despite adverse consequences, a process closely linked to dopamine-induced changes in the Nucleus Accumbens (NAc) and its role as the brain's "reward center." The NAc integrates information from various brain regions, including the Paraventricular Thalamus (PVT), to produce motivated behaviors. Recent studies have identified the PVT, especially its anterior segment (aPVT), as a critical hub in addiction neurocircuitry, but findings have been inconsistent, likely due to the PVT's heterogeneity and the specific neurochemical and anatomical properties of its connections to the NAc. Prior research has shown that aPVT neurons, identifiable by neurotensin expression, send excitatory projections to the NAc, which are modulated by endogenous cannabinoids (eCBs). These interactions suggest a complex regulatory mechanism. Preliminary experiments used techniques including transsynaptic viral tracing and in vivo calcium imaging, to study the activity dynamics of NAc neurons, particularly those expressing Proenkephalin (PENK) and receiving aPVT inputs, during reward-seeking tasks. I propose to extend these findings by employing a multidisciplinary approach that combines experimental neuroscience with sophisticated computational analysis. By applying dimensionality reduction techniques, clustering algorithms, and machine learning models to neural and behavioral data, I aim to map the functional connectivity within the NAc and elucidate the roles of specific neuronal ensembles in reward-seeking behavior. This comprehensive analysis will not only clarify the neurobiological underpinnings of addiction but also contribute to the development of targeted therapies for addiction and related disorders, leveraging the unique intersection of computational neuroscience and behavioral analysis.

The nucleolus is an essential subnuclear organelle that performs central regulatory roles in cellular metabolism, epigenetic programming, and stress signaling. In mammals, nucleoli are disassembled and rebuilt de novo with each cell division, through an elaborate assembly mechanism that has long eluded molecular characterization. This assembly process is spatiotemporally controlled by a long noncoding RNA termed the 47S pre-ribosomal RNA (47S pre-rRNA), which initiates nucleolar assembly at the site of its transcription, and for which continued expression is required to maintain nucleolar integrity. Yet, while the 47S’ roles in nucleating and scaffolding nucleolar architecture are well established cytologically (they were first observed nearly a century ago), the structural elements on the 47S that enable these architectural functions remain unknown. I hypothesize that an RNA domain within the 47S, termed the 5´–External Transcribed Spacer (5´–ETS), harbors the long-sought structural scaffolds of the nucleolus. To test this, I am implementing a live-cell reporter assay that will monitor, in real time, if transcripts derived from the 5´–ETS drive nucleolar localization and architecture. My approach leverages recent advancements in artificial gene synthesis and live-cell RNA imaging. A novel drug-inducible promoter will enable me to temporally control expression of 5´–ETS sequence variants in live cells. I will monitor the kinetics and efficiency with which these transcripts localize into the nucleolus by two-color live cell imaging, using the newly discovered fluorescent RNA aptamer RhoBAST, and a fluorescently tagged nucleolar marker protein. To design our negative controls, I implemented a bioinformatic pipeline that generates scrambles of long, low-complexity RNA sequences—ablating primary structure but preserving dinucleotide content. This allows us to investigate whether nucleotide composition or sequence affects nucleolar formation. We anticipate that this powerful system will set the stage for detailed molecular characterization studies, revealing the long-elusive molecular interactions that control nucleolar architecture in health and disease.

Cannabis use has dramatically increased in response to legalization in the U.S., with U.S. sales jumping 46% from 2019 to 2020. ᐃ9-tetrahydrocannabinol (THC) is the primary psychoactive compound in Cannabis, and it has been shown to modify learning and motivation amongst regular users. Learning and motivation are key central processes primarily organized by the prefrontal cortex (PFC) brain region. I sought to test effects of THC on PFC activity during appetitive Pavlovian conditioning in mice- a behavior in which a subject learns to pair two stimuli together over time. Doing so provided much needed insight into learning and motivation under the effect of THC. THC acts on the endocannabinoid CB1 receptor, a presynaptic signaling protein responsible for modulating neural activity throughout the brain, with robust expression in the PFC. To monitor neural activity during behavioral trials, we implanted optic fibers into the PFC and virally expressed biological sensors. We used VGLUT1-Cre mice with a Cre-dependent GCaMP6f sensor to selectively target pyramidal glutamatergic activity during conditioning. We also utilized machine learning tracking software, SLEAP, to analyze behavior through video recordings. In our conditioning paradigm, animals were presented with a houselight and a sucrose reward, which they consolidated an association between after many trials. The mice experienced 5 days of Pavlovian conditioning, and I injected a moderate i.p. dose of THC (5 mg/kg) to one cohort, while another was given a vehicle before undergoing further trials. Our preliminary results showed that glutamatergic activity correlated with learning and association to the cue over time. We expected and observed that THC decreased the signals across the animals and reduced motivation. We categorized THC-induced behavior using SLEAP, a program tracking the mouse’s body parts to capture real-time movement, and found that locomotion decreased and resting behaviors increased in the THC cohort.

A key neuromodulatory system involved in anxiety disorders is the locus coeruleus noradrenergic system (LC-NE), which projects broadly throughout the central nervous system. The LC is stress responsive and tonic activation of the LC and its projections to the BLA is anxiogenic. Previously, the Bruchas Lab has used two-photon calcium imaging to show that a powerful stressor (predator odor) increased synchronous activity of LC neurons. They also found that mimicking this predator odor evoked activity with optogenetics altered the activity of individual neurons downstream in the BLA in a β-adrenergic receptor (β-AR) dependent manner. Although these data support the LC's involvement in promoting aversion and increasing anxiety-like behavior, the specific neurotransmitter, neuronal cell types, and receptors responsible for these effects remain unidentified. Therefore in hopes of identifying these specific signaling molecules and neuronal cell types and receptors, I first used fiber photometry and a novel biosensor (GRABNE2m) to detect norepinephrine (NE) release in the BLA while mice were exposed to a predator odor. I found that predator odor produced robust increases in NE release in the BLA compared to control odor (n=5, 3 male, 2 female) Further, we found that optogenetic activation of terminals from the LC to the BLA produced very similar levels of NE release compared to what was evoked by predator odor. To determine the cell type and receptor that is sensing this stress-induced NE release, I used a CRISPR/SaCas9 virus, developed in collaboration with Dr. Larry Zweifel’s lab, to knock-down β2-adrenergic receptors (β2-ARs) in glutamatergic BLA neurons to test their causal role in stress-induced anxiety-like behavior. CRISPR knockdown of β2-ARs in the BLA blocked several stress-induced anxiety-like behaviors (n=4, 4 female). By understanding the circuit-based mechanisms of how stress-induced anxiety is regulated, researchers could identify potential targets for therapeutic treatments of anxiety disorders.

Cannabidiol (CBD), a non-psychoactive cannabinoid compound found in cannabis, has been reported to attenuate morphine tolerance and can potentially be used as an alternative to opioids in treating chronic pain. Previous work has established connections between morphine tolerance and Reactive Oxygen Species (ROS) production through JNK-mediated Peroxiredoxin 6 (PRDX6) activation. Excess ROS production promotes desensitization of opioid receptors, which in turn leads to opioid tolerance. CBD administration is associated with decreasing pain-related Reactive Oxygen Species (ROS) production, and it was hypothesized that CBD directly interacts with JNK, blocking JNK’s activities. This project aims to investigate the connections between CBD administration and ROS production to determine CBD’s effects on JNK-mediated ROS production. To quantify ROS production through fluorescence imaging, I will transfect wild-type HEK 293 cells with oROS, a genetically encoded sensor, which fluoresces proportionally to ROS production. Coverslips of HEK 293 cells expressing oROS are treated with buffer (control) and CBD before administration of Tumor Necrosis Factor alpha (TNFα), a known activator for JNK released during pain states. After imaging with oROS, I will quantify ROS production and compare this between groups with and without CBD pretreatment to determine CBD’s activity on inhibiting JNK-mediated pro-inflammatory pathways. I predict that relative to the control, cells treated with CBD will have significantly less ROS production. If the results are consistent with this prediction, CBD could be a potentially promising co-treatment with opioids in managing chronic pain as it can potentially attenuate opioids' side effects like tolerance. 

The peptide neurotensin (NTS) has been known as a regulator of dopamine neuron activity and its system, which modulates numerous functions in the brain. Although ample research has now demonstrated that NTS in Ventral Tegmental Area (VTA) increases dopamine release in some regions, much remains unknown about the endogenous sources of NTS in the VTA and the impact of physiological NTS release. Recent NTS mapping data from the Soden lab demonstrated that there is a NTS projection from Periaqueductal Gray (PAG) to the VTA and also to hindbrain regions including the ventral medulla. This project investigates the effect of this interconnection between these three regions on the dopamine system. Utilizing advanced techniques in circuit mapping, mice will be injected in the VTA and the ventral medulla with fluorescent Retrobeads or a retrograde virus (rAAV2 anti mcherry or GFP), which are taken up by synaptic terminals and migrate up the axon retrogradely to label cell bodies, one color assigned for each region. Following euthanasia, mice brains will undergo immunohistochemistry such as histology and in-situ RNA staining. Then, data will be collected using imaging microscopes for results and further analysis. If NTS neurons in the PAG have green and red expressions, this will indicate that the same population of neurons send axons to both downstream regions, compared to PAG NTS neurons with only one color, indicating the presence of two separate neuron populations. Experiments on retrograde mapping of NTS inputs will contribute to building onto our current knowledge about VTA-PAG-ventral medulla circuit and effects on dopamine neurons following their interplay. In the end, our goal is to establish a novel understanding of endogenous NTS signaling mechanisms, mediation of complex reward processes, and treatment targets with experimental outcomes, giving rise to the development of therapeutic interventions towards addiction and related psychiatric disorders.

Activation of Kappa Opioid Receptors (KOR)- either from a stress-evoked release of the endogenous dynorphin neuropeptide or pharmacologically- produces analgesic effects, aversive stress responses, and amplifies behaviors related to drug addiction. Influence on these specific behaviors can be attenuated through naloxone precipitated fentanyl withdrawal to model extreme distress. This was replicated by surgically implanting osmotic minipumps filled with fentanyl in mice for a 7-day period. Saline was utilized as a control against mice pretreated with norBNI (a long-lasting KOR antagonist) versus mice who only received fentanyl in order to determine if there was an effect on behavioral response following the precipitated withdrawal. Once the pumps were removed and fentanyl was eliminated from the system, mice underwent a 2-day spontaneous withdrawal phase prior to pairing 1 mg/kg naloxone injections with the presentation of almond extract. An observed aversion response to the almond odorant would exhibit a conditioned stimulus. The pairing of these components would then associate the negative feelings from withdrawal with introduced extract. The odorant aversion evident in fentanyl-treated mice was significantly reduced by pretreatment with the KOR antagonist norBNI, suggesting that the aversion was mediated by the release of endogenous dynorphin. With the continuation of this experiment, I would expect to see an increase in stress resilience as the KOR system becomes blocked with the administration of an antagonist.

Understanding the complex neural mechanisms underlying motivated behavior and reward processing is crucial for advancing our knowledge of addiction and related disorders. In this study, we explore the role of neurotensin (NTS) peptides in modulating dopamine neurons within the ventral tegmental area (VTA), a key brain region implicated in reward processing. Previous research has highlighted the reinforcing effects of optogenetic stimulation of NTS inputs from the lateral hypothalamus to the VTA. However, a gap remains in understanding how NTS signaling interacts with other brain regions involved in motivated behavior. Our research addresses this gap by investigating the contribution of NTS and fast neurotransmitters in circuit-specific behavioral activation within the VTA. We hypothesize that distinct NTS inputs differentially influence motivated behavior in a combined NTS and GABA/glutamate-dependent manner through optogenetics and circuit mapping. We employ a multidisciplinary approach combining optogenetics, CRISPR gene editing, and behavioral assays to achieve this. Specifically, I utilize optogenetic stimulation to selectively activate NTS inputs from diverse brain regions, including the periaqueductal gray (PAG) and the pedunculopontine tegmentum (PPTg), targeting specific input regions via unilateral injections of AAV1-FLEX-Chrimson-mCherry. Control mice receive AAV1-FLEX-mCherry alone to ensure the specificity of our manipulations. Following surgical procedures and acclimation to the experimental setup, I conduct behavioral assays on mice, specifically Real-Time Place Preference (RTPP) and open-field testing to assess the effects of optogenetic stimulation on locomotion and anxiety-related behaviors. Subsequently, we investigate the mediating role of NTS and neurotransmitters by employing CRISPR viruses to target genes related to different components of neurotransmission. By integrating cutting-edge techniques with behavioral assessments, our study aims to elucidate the intricate interplay between NTS signaling, fast neurotransmitters, and specific circuit activations within the VTA. These findings contribute to our understanding of reward processing and hold potential implications for developing novel addiction treatment strategies.

The Kappa Opioid Receptor (KOR) is one of four opioid receptors found in the body and has an endogenous ligand known as Dynorphin. Binding of Dynorphin to this receptor has been shown to mediate some responses to stress, increase addiction risk, affect learning behaviors, and can provide analgesic effects. I am interested in learning how stress-induced activation of the dynorphin-KOR system affects addiction risk by identifying where in the brain dynorphin acts and how KOR activation affects brain function. I studied "where" by doing injections into specific regions in which Dynorphin is expressed and observing the mechanism of activated KOR. As a part of my project, I was tasked with performing stereotaxic viral infections of two different viruses in order to further study this system and its pathways. To allow for selective targeting of KOR expressing cells, the mice used have a gene for Cre Recombinase placed after the KOR promoter. This creates an environment where all neurons with expressed KOR also have Cre Recombinase in the cytoplasm. From here, I inject double-floxed inverse orientation viral vectors into specific KOR-expressing regions of the brain. The reversed virus is able to find its way into many cells but is only able to be inverted and properly expressed in cells containing Cre Recombinase. Functions of the active virus may differ but there are two primary examples used in my projects. The first is p38 CRISPR, which uses CRISPR-Cas9 technology to knockout the p38 mitogen-activated protein kinase gene, a protein kinase expressed after KOR activation. The second is a reactive oxygen species sensor, as ROS are associated with depalmitoylation and subsequent deactivation of the receptor. The use of a stereotax with this technology allows for precise targeting of different brain regions depending on the location relative to landmark sutures on the mouse skull.

 âˆ†9-Tetrahydrocannabinol (THC), the primary psychoactive compound in Cannabis, is responsible for the experience known colloquially as “being high.” Considering its alarmingly high rates of usage, THC’s effects on movement behavior are insufficiently studied. My project addresses this crucial gap in our knowledge by investigating the dose-dependent effects of THC on movement behavior using mouse models in tandem with novel behavioral neuroscience techniques. My research aims to establish a preclinical model for THC-induced impairment, focusing on studying its impact on locomotor control. My main experimental tool is a behavioral linear track, which is a clear glass corridor with a 45 degree-angled mirror placed beneath it. The linear track allows us to create a standardized multi-dimensional environment in which mice are recorded after they are treated with either a control or variable doses of THC. The videos taken of the mice are then analyzed using SLEAP. SLEAP is a machine-learning, pose-estimation algorithm that I helped train to track individual points of interest on the mice, such as the nose, paws, and tail. Behaviors of interest, such as walking, rearing, and grooming, are classified by a random forest algorithm that analyzes SLEAP label data to output identified behaviors. This data is then tabulated and graphed to reflect the dose-dependent changes in behavior elicited by THC. These classifications are also used to further analyze metrics during a represented behavior. For instance, for a walk event, we can utilize positional data from SLEAP to calculate and measure kinematic features such as stride length and limb speed, allowing us to distinguish between an unimpaired and an impaired walk. This computerized analysis approach minimizes human bias, reduces error, and produces exhaustive data that can characterize subtle differences in behavior, like when comparing mice exposed to low THC doses of 0.1mg/kg and 0.3 mg/kg.

Thousand-and-one amino acid kinase 2 (TAOK2) is an autism-associated serine-threonine kinase that has been shown to be important for several aspects of neurodevelopment, including axon elongation, dendritic branching, and spine formation. TAOK2α is localized to the endoplasmic reticulum (ER) and mediates the tethering of the ER to the microtubule cytoskeleton. Interestingly, it is found in specific subdomains within the ER membrane, however, the identity and function of these distinct ER subdomains remain unknown. Further, during mitosis, TAOK2α is found at the points of contact between ER and mitotic spindles including mitotic spindle poles or centrosomes. While it is clear that TAOK2 plays an important role in regulating ER dynamics during cell division, the molecular mechanisms mediating these functions and specific localization of TAOK2 are yet to be determined. To better understand the molecular function of TAOK2, I performed an immunoprecipitation (IP) mass spectrometry in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) and identified 31 potential interactors of TAOK2α. These interactors are implicated in several important cellular processes such as mitosis, RNA splicing, transcription and translation, as well as lipid metabolism. I will further shortlist the interactors by validating the interaction through immunocytochemistry. I will examine changes in cell cycle in wildtype and TAOK2 knockout NPCs through live cell confocal microscopy. Additionally, given that some of the interactors regulate lipid homeostasis, I performed lipidomics to study how the absence of TAOK2 affects lipid metabolism. Together, these experiments will reveal fundamental insights on TAOK2 and the unique link between dysfunction in organelles and autism.

TBC1(Tre2/Bub2/Cdc16) Domain-Containing Kinase (TBCK) is a pseudokinase with proposed involvement in the endocytic pathway. Kinases are proteins that can post-translationally modify other proteins through the addition of inorganic phosphate from ATP to serine/threonine/tyrosine residues. TBCK, being a pseudokinase, lacks critical residues that allow ATP binding and, therefore, cannot catabolize ATP. Pseudokinases, while catalytically inactive, have been shown to have protein scaffolding properties as well as modulate the activity of other kinases. Whether pseudokinase TBCK plays a role in any of those functions has yet to be discovered. Via the TBC1 domain, TBCK interacts with Rab proteins, a class of membrane-binding proteins involved in multiple cellular pathways that coordinate intracellular vesicle transport with GTP active and GDP inactive states. TBCK functions as a Rab GAP(GTP-hydrolysis activating protein), hydrolyzing Rab bound GTP and leaving an inactive GDP-bound Rab. Mutations in TBCK have been found to be clinically associated with a rare neurological disorder, TBCK syndrome, characterized by delayed development, intellectual disorder, and hypotonia. The interactors and Rab substrates of TBCK are under researched and still poorly understood; we aim to illuminate those interactions through immunoprecipitation (IP) and mass spectrometry. Early attempts at this goal involved co-transfection of various Rab protein targets with TBCK WT and TBCK R511H (a TBC1 inactive mutant) in HEK 293T cells and subsequent co-IP, enriching for TBCK and interacting Rab proteins. These preliminary results, in combination with live imaging and immunofluorescence of TBCK and its mutants with Rab proteins and other membrane markers, proved inconclusive. Therefore, we are now performing crosslinking immunoprecipitation mass spectrometry to allow the identification weakly interacting protein complexes through mass spectrometry. These experiments will provide insight into the fundamental biology underlying TBCK’s role in neurodevelopment and how its dysfunction contributes to disease states.

Various anti-cancer therapeutics, known as microtubule targeting agents (MTAs), target the microtubule, a tube-like structure that is a major component of cell mechanisms including mitosis and maintenance of the cell shape. MTAs selectively bind to tubulin — the building block of microtubule —, disrupting microtubule dynamics and inducing cell death. Despite their known impact on antitumor activity, the precise mechanism by which MTAs promote cell death remains unclear. To understand the efficacy of ST-401 (an MTA drug) as a tumor suppressor, I conduct various assays including drug treatment and Western blot to compare the expression of specific proteins in response to the drug treatments. These assays contribute to understanding cellular processes, molecular interactions, and the effects of various treatments or conditions on cells. Currently, I am being trained to conduct an experiment called XFe Seahorse analyzer. I’m carrying out this experiment to assess how the laboratory-discovered drug affects the mitochondrial function in various cancer cell lines. This experiment aims to determine whether the compound down-regulates mitochondrial function, leading to cell death. I’m leading a project to test a specific fission (cell splitting) protein, DRP1, and how its protein level responds to treatment with ST-401 in two GBM cell lines (resistant and sensitive) and one Colon cancer cell line. DRP1 regulates mitochondrial fission to maintain healthy mitochondrial function. Recently, I found an increase in mitochondrial fission 24 hours after ST-401 treatment in the sensitive GBM cell line, so I’ll further examine DRP1 expression by drug treatment and Western blot to understand these results and see whether ST-401 recruits DRP1, resulting in promotion of mitochondrial division. After DRP1 project, I will expand the project to asses the combination drugtreatment where we combine FDA-approved cancer drug and ST-401 to reflect real-world scenarios, aiming to ensure clinical relevance and safety by studying potential drug interactions.

BK channels are potassium channels activated in response to depolarization and elevated intracellular calcium ion levels. It has been observed that BK channels form clusters in cells, but the mechanism for clustering has not been characterized. This project attempts to discover important components that lead to BK channel clustering using super-resolution microscopy, proximity ligation assay, and Fluorescence Recovery After Photobleaching (FRAP) experiments. One possible mechanism relates to denser regions in the plasma membrane of PtdIns(4,5)Pâ‚‚ as a possible lipid raft, hypothesized to localize proteins. We used tsA-201 cells to express BK channels (α subunit). In FRAP experiments, BK channels were tagged with a green fluorescent protein (GFP). To modify the levels of PtdIns(4,5)Pâ‚‚, we co-expressed PIP5Kγ, the enzyme that catalyzes the synthesis of PtdIns4P to PtdIns(4,5)Pâ‚‚. Expression of PIP5Kγ is known to increase PtdIns(4,5)Pâ‚‚ levels by 30%. In our analysis, we assumed that large, bright fluorescent dots in live cells correspond to BK clusters. We found that co-expression of PIP5Kγ with BK decreases cluster size by 43% in super-resolution experiments and increases the number of puncta (BK clusters) by 41% in PLA experiments. FRAP experiments on a PIP2 biosensor, PH-PLCδ1-GFP, showed reduced fluorescence recovery speed when PIP5Kγ was co-expressed. Future FRAP experiments observing BK channels will allow us to determine if membrane components, such as PtdIns(4,5)Pâ‚‚, influence the integrity and mobility of BK clusters and if the addition of these lipids is sufficient to induce additional cluster formation.