Adipose tissue, also known as body fat, is vital in storing energy. It is composed of adipocytes and present in different depots. In this study we focused on omental (OM) adipose tissue, which is found between organs near the stomach, and subcutaneous (SC) adipose tissue, which is found under the skin. Functional differences have been observed among adipose depots. SC adipose tissue is responsible for insulation while OM adipose tissue has endocrine functions. OM adipocytes have been observed to be smaller and more variable in size compared to SC adipocytes in individuals with obesity. I hypothesized that the size of both OM and SC adipocytes is associated with increasing BMI. I tested this hypothesis using SC and OM adipose tissue biopsies collected during elective surgeries from metabolically healthy participants (20 females, 11 males) with a range of ages (25-65 years) and BMIs (21-56). Afterwards, the tissues were fixed and stained with H&E. I drew 2-5 squares per slide and counted the number of adipocytes within each square. The size difference between OM and SC adipocytes was tested using a Wilcoxon signed-rank test and a significant difference (p=0.004) was observed. A correlation between BMI and the size of OM (p = 0.008) or SC (p = 0.009) adipocytes was detected with weighted linear regressions. Sex was not observed to be a significant covariate. These findings expand on prior data by including lean individuals, and patients with obesity who are otherwise metabolically healthy. The results show there is a clear difference in the size of adipocytes. The adipocyte size in both depots correlated with BMI. This data shows that progressive obesity and adipose tissue enlargement is due to the enlargement of the adipocytes rather than an increase in the number of adipocytes in both OM and SC depots.

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.

 Thousand and one amino acid kinase 1 (TAOK1) is a serine/threonine kinase expressed abundantly in neurons in the mammalian brain. Mutations in TAOK1 are closely associated with both autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD). Studies have shown that TAOK1 is a component of the Hippo pathway, which modulates cell proliferation, apoptosis, and differentiation. Loss of TAOK1 in mice causes decreased neuronal migration and defects in dendritic development, however, mechanisms that lead to neuropathology in patients with TAOK1 mutations are not known. We focused our investigation on a patient mutation in TAOK1, S111F, which clinically manifests as intellectual disability, developmental delay, and macrocephaly. We used induced pluripotent stem cell (iPSC) derived 3D cortical brain organoids with heterozygous and homozygous S111F mutation as a model system in order to examine phenotypical changes in early brain development. We also grew 2D cultures of neural progenitor cells (NPCs), and I used Fiji image analyses to track proliferation rates, which were significantly increased in the mutant cells compared to isogenic control. Western blots were used to observe the phosphorylation states of proteins in the Hippo pathway between wild-type and mutation-carrying cell lines. These experiments show that TAOK1 kinase-dead mutation led to shut down of the Hippo pathway, as evidenced by nuclear accumulation of Yap-Taz proteins. In a parallel research direction, the Yadav lab is interested in solving the structure of TAOK1 membrane binding region. To this end, I am using E. coli bacteria to purify TAOK1 protein using a bacterial expression vector, and after protein purification, the purified protein will be used in biochemical and structural studies. These studies will provide mechanistic insight into TAOK1 biology and how patient mutations in TAOK1 contribute to neurodevelopmental disorders.