JI WOO PARK
Defining host and viral protein involvement in mitochondrial remodeling during infection
Mitochondria are dynamic organelles that control the cell’s energy metabolism, immune signaling, and lifespan. In response to various stimuli, including viral infections, mitochondria are remodeled, displaying alterations in their shape, distribution, abundance, as well as ultrastructure. These remodeling events often involve intra- and inter-organelle contact sites, such as between mitochondria and the ER. Although the field has a good understanding of the mechanisms controlling mitochondrial fusion and fission, the knowledge of mitochondrial structure-function relationships and multi-organellar structures in the context of an infection remains limited. I will use infection with human cytomegalovirus (HCMV) as a model system for my studies of intra- and inter-organellar mitochondrial remodeling events. HCMV infection induced pronounced mitochondrial fragmentation, while surprisingly also elevating mitochondrial bioenergetics. Prior findings from our group lead me to hypothesize that HCMV leverages a combination of host and viral factors to regulate mitochondrial ultrastructure and mitochondria-ER interactions with fine temporal specificity. To address my hypothesis, I will integrate molecular virology with microscopy, proteomics, and metabolomics analyses. In Aim 1, I will establish the mechanism of mitochondria-ER encapsulation (MENC) formation and its temporal functions during HCMV infection. MENC is a unique proviral structure uncovered by our lab in which a mitochondrion is stably and asymmetrically encapsulated by the ER, thereby stabilizing their interaction and increasing mitochondrial membrane potential. My preliminary data show that loss of outer mitochondrial membrane protein MFN2 promotes MENC formation and suppresses viral replication, thus suggesting that MENC’s functions are temporally controlled during HCMV infection. In Aim 2, I will elucidate how the HCMV viral protein pUL13 alters mitochondrial ultrastructure and membrane contact sites (MCS). Our lab has previously shown that pUL13 localizes to the mitochondria during HCMV infection and is required for the increase in oxidative phosphorylation (OXPHOS) to support viral replication. I hypothesize that pUL13-dependent upregulation of OXPHOS is achieved through compaction of mitochondrial cristae and modulation of mitochondrial MCS, thus affecting MENCs and mitochondria-mitochondria contact sites (MiMiCS).
Recording Developmental Trajectories in Trunk-Like Structures using Single-Cell Molecular Barcoding
Understanding the differentiation events and developmental trajectories that occur during embryogenesis is one of the key questions of developmental biology. However, studying the mechanisms that govern these decisions remains challenging due to the poor scale and tractability available when working in vivo. To overcome this, recent advances in in vitro gastruloid systems have provided the opportunity to study mammalian development at resolutions and scales that are not yet possible in vivo. While these systems offer great ability to perturb and study developmental processes, it is not yet fully understood how faithfully these systems recapitulate in vivo pathways, limiting the biological conclusions that are possible with these systems. To address this, working with Adriano Bolondi from the Meissner lab and Zachary Smith at Yale, we have developed a platform that uses a CRISPR/Cas9 based lineage tracer to study developmental trajectories in Trunk-Like Structures (TLS), an advanced gastruloid that models the posterior development of mouse gastrulation. In a proof-of-concept experiment, we have shown that this platform can be used to reconstruct high resolution lineage relationships in TLS, and that the population of neuromeosdermal progenitors (NMPs) follows similar trajectories to their in vivo counterparts. While these datasets provide great insight into the general trajectories present in TLS, higher numbers of replicates are needed to develop more refined analysis methods. Building on this work, I propose to leverage this platform to investigate variation across Trunk-Like Structures. We will generate a large TLS dataset from 96 separate structures and construct a computational pipeline to carry out the multiplexed single-cell RNA analysis and tree reconstruction. Then, using these datasets, I will develop analytical methods to investigate the consistency of developmental programs that occur across TLS replicates. Finally, to better understand the similarities with in vivo development, I will compare the TLS datasets against available single-cell datasets of in vivo development. By integrating TLS and in vivo datasets, I will interrogate the differences in cell signature and composition between in vivo and TLS progenitor populations. Taken together, this project will create a platform that will allow for more in depth investigation of TLS development and plasticity, as well as strengthen the biological claims that can be made from these systems.
Petry and Stone Labs
Toward microtubule driven micro-robot
Micro-robot is a promising technology with the potential to be used in various fields such as drug delivery, water purification, and so on. Rapid progress in nanoscale bioengineering has allowed for the design of biomolecular devices that act as sensors, actuators, and even logic circuits. Realization of micrometer-sized robots assembled from these components is one of the ultimate goals of bioinspired robotics. Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can generate force during their polymerization; thus, microtubule has high potential as an actuator for changing the shape of a micro-robot. However, there is still a lack of research on how to uniformly generate microtubule driven micro-robots and quantitative analysis of their dynamics. I will use the microfluidic system to uniformly generate a micro-robot consisting of a vesicle made of lipid bilayer and xenopus egg extract and analyze their actuating motion according to the size of the robot and the concentration of Ran which is a protein regulate the microtubule nucleation.