Phillip Kyriakakis, Ph.D. is Senior Research Scientist in the Bioengineering Department at Stanford University in the Wu Tsai Institute for Neuroscience. Dr. Kyiakakis did his undergraduate work in Biochemistry at UMass Boston where he also worked in the lab of Dr. Alexey Veraksa's developmental biology lab doing biochemical experiments as well as starting to develop PhyB optogenetics (2008). Dr. Kyriakakis continued his education at UC San Diego in the Division of Biological Sciences. There he studied cellular programming and metabolism to obtain his degree with a specialization in Multiscale Biology. Dr. Kyriakakis did his postdoctoral work in the Bioengineering Department at UC San Diego with Todd Coleman continuing the development optogenetic tools and related technologies. In 2021 Dr. Kyriakakis moved to his Senior Research Scientist role at Stanford University in the Bioengineering Department in the Wu Tsai Institute for Neurosciences.
I am interested in building tools to study biology. In the process of engineering, it is often the case that a better understanding of the system is needed to build it or to optimize new tools. Thus, tool building at the cutting edge is often a combination of exploration and engineering, a combination that I find very exciting. My interests are to develop tools with capabilities far beyond what exists today and to apply them to understand important biological phenomena. Further, I aim to make these tools as accessible as possible to other researchers that want to use them or improve on them. I enjoy being a part of the scientific community where we can work together openly to maximize the impact of our work.
My specific interests constantly change as new challenges arise. Starting by developing new optogenetic tools for controlling genes or other biological processes, I found bottlenecks to apply them more broadly, (1) delivering large genes or multipart genetic circuits and (2) assembling these genetic circuits (3) the need for low-cost illumination systems. To improve these, our team is developing (1) novel viral systems and (2) DNA assembly systems. In addition, we developed an inexpensive ($200-$700) computer controllable LED illumination system with detailed step-by-step instructions so that it can be built without prior experience in building electronics (https://www.jove.com/t/61914). In addition to these projects, we are also developing automated platforms for rapid customization and optimization of these tools.
Lipopolysaccharide-induced maternal immune activation modulates microglial CX3CR1 protein expression and morphological phenotype in the hippocampus and dentate gyrus, resulting in cognitive inflexibility during late adolescence.
Brain, behavior, and immunity
Inflammation during pregnancy can disturb brain development and lead to disorders in the progeny, including autism spectrum disorder and schizophrenia. However, the mechanism by which a prenatal, short-lived increase of cytokines results in adverse neurodevelopmental outcomes remains largely unknown. Microglia-the brain's resident immune-cells-stand as fundamental cellular mediators, being highly sensitive and responsive to immune signals, which also play key roles during normal development. The fractalkine signaling axis is a neuron-microglia communication mechanism used to regulate neurogenesis and network formation. Previously, we showed hippocampal reduction of fractalkine receptor (Cx3cr1) mRNA at postnatal day (P) 15 in male offspring exposed to maternal immune activation induced with lipopolysaccharide (LPS) during late gestation, which was concomitant to an increased dendritic spine density in the dentate gyrus, a neurogenic niche. The current study sought to evaluate the origin and impact of this reduced hippocampal Cx3cr1 mRNA expression on microglia and cognition. We found that microglial total cell number and density are not affected in the dorsal hippocampus and dentate gyrus, respectively, but that the microglial CX3CR1 protein is decreased in the hippocampus of LPS-male offspring at P15. Further characterization of microglial morphology in the dentate gyrus identified a more ameboid phenotype in LPS-exposed offspring, predominantly in males, at P15. We thus explored maternal plasma and fetal brain cytokines to understand the mechanism behind microglial priming, showing a robust immune activation in the mother at 2 and 4 hrs after LPS administration, while only IL-10 tended towards upregulation at 2 hrs after LPS in fetal brains. To evaluate the functional long-term consequences, we assessed learning and cognitive flexibility behavior during late adolescence, finding that LPS affects only the latter with a male predominance on perseveration. A CX3CR1 gene variant in humans that results in disrupted fractalkine signaling has been recently associated with an increased risk for neurodevelopmental disorders. We show that an acute immune insult during late gestation can alter fractalkine signaling by reducing the microglial CX3CR1 protein expression, highlighting neuron-microglial fractalkine signaling as a relevant target underlying the outcomes of environmental risk factors on neurodevelopmental disorders.
View details for DOI 10.1016/j.bbi.2021.07.025
View details for PubMedID 34343619
Building a Simple and Versatile Illumination System for Optogenetic Experiments
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.
View details for DOI 10.3791/61914
View details for Web of Science ID 000646171700033
View details for PubMedID 33522514
Engineering light-controllable CAR T cells for cancer immunotherapy
2020; 6 (8): eaay9209
T cells engineered to express chimeric antigen receptors (CARs) can recognize and engage with target cancer cells with redirected specificity for cancer immunotherapy. However, there is a lack of ideal CARs for solid tumor antigens, which may lead to severe adverse effects. Here, we developed a light-inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control CAR T activation. We first demonstrated light-controllable gene expression and functional modulation in human embryonic kidney 293T and Jurkat T cell lines. We then improved the LINTAD system to achieve optimal efficiency in primary human T cells. The results showed that pulsed light stimulations can activate LINTAD CAR T cells with strong cytotoxicity against target cancer cells, both in vitro and in vivo. Therefore, our LINTAD system can serve as an efficient tool to noninvasively control gene activation and activate inducible CAR T cells for precision cancer immunotherapy.
View details for DOI 10.1126/sciadv.aay9209
View details for Web of Science ID 000514842000030
View details for PubMedID 32128416
View details for PubMedCentralID PMC7030928
An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics
ACS SYNTHETIC BIOLOGY
2019; 8 (10): 2359-2371
While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.
View details for DOI 10.1021/acssynbio.9b00175
View details for Web of Science ID 000492115800019
View details for PubMedID 31592660
View details for PubMedCentralID PMC8135225
Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP(+) Reductase Systems Enables Genetically Encoded PhyB Optogenetics
ACS SYNTHETIC BIOLOGY
2018; 7 (2): 706-717
Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
View details for DOI 10.1021/acssynbio.7b00413
View details for Web of Science ID 000426012600042
View details for PubMedID 29301067
View details for PubMedCentralID PMC5820651
Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.
View details for DOI 10.3791/55804
View details for Web of Science ID 000406213900084
View details for PubMedID 28605365
View details for PubMedCentralID PMC5608175
Heparin Mimicking Polymer Promotes Myogenic Differentiation of Muscle Progenitor Cells
2010; 11 (12): 3294-3300
Heparin and heparan sulfate mediated basic fibroblast growth factor (bFGF) signaling plays an important role in skeletal muscle homeostasis by maintaining a balance between proliferation and differentiation of muscle progenitor cells. In this study we investigate the role of a synthetic mimic of heparin, poly(sodium-4-styrenesulfonate) (PSS), on myogenic differentiation of C2C12 cells. Exogenous supplementation of PSS increased the differentiation of C2C12 cells in a dose-dependent manner, while the formation of multinucleated myotubes exhibited a nonmonotonic dependence with the concentration of PSS. Our results further suggest that one possible mechanism by which PSS promotes myogenic differentiation is by downregulating the mitogen activated extracellular regulated signaling kinase (MAPK/ERK) pathway. The binding ability of PSS to bFGF was found to be comparable to heparin through molecular docking calculations and by native PAGE. Such synthetic heparin mimics could offer a cost-effective alternative to heparin and also reduce the risk associated with batch-to-batch variation and contamination of heparin.
View details for DOI 10.1021/bm101041f
View details for Web of Science ID 000285267500009
View details for PubMedID 21058638
View details for PubMedCentralID PMC4136807
beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development
2010; 29 (19): 3222-3235
β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz.
View details for DOI 10.1038/emboj.2010.202
View details for Web of Science ID 000283700700003
View details for PubMedID 20802461
View details for PubMedCentralID PMC2957207
Tandem affinity purification in Drosophila - The advantages of the GS-TAP system
2008; 2 (4): 229-235
Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.
View details for DOI 10.4161/fly.6669
View details for Web of Science ID 000259006400007
View details for PubMedID 18719405