Research Projects

Compartmentalization is necessary for the efficiency and regulation of the many biochemical interactions occurring in living cells. Well-known are the various eukaryotic organellar structures that are bounded by phospholipid membrane bilayers. However, more recently established are the numerous membraneless organelles (MLOs) found within the cytoplasm, nucleus and mitochondria that are thought to result from liquid-liquid phase separation (LLPS) which is the spontaneous partitioning into dense and dilute phases due to favorable interactions between the separating molecules. These MLOs are “liquid-like bodies”, typically rich in nucleic acids and intrinsically disordered proteins (IDPs). IDPs tend to form insoluble aggregates, which can be promoted via numerous physicochemical perturbations. The primary hypothesis is that the IDPs first undergo LLPS to form liquid-like granules which then transform into solid-like aggregates. These aggregates serve as precursors of various neurological diseases. In vitro studies reveal that IDPs undergo LLPS to form liquid condensates (droplets) through low-affinity interactions among disordered regions, without a need for any additional cofactors. However, in cell, this process is far more complicated due to the presence other interactions. Proposed work will use TAU protein which undergoes phase separation at micromolar concentrations to form liquid condensates in vitro, and they mature over time into multiple solid-like forms. This in vitro model of both phase separation and maturation will be used as a methodological test bed. TAU protein is a member of the microtubule associated protein (MAP) family, which stabilizes and regulates axonal microtubules in central nervous system, is a key protein involved in many neurodegenerative diseases including Alzheimer’s disease, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and many more. Brains of the patients suffering from these diseases contain solid aggregates enriched in TAU. Healthy cells, however, corroborate that the LLPS-based compartments are disassembled before they mature through multiple mechanisms among which one is by using chaperones. An important clue towards understanding the formation of these aggregates is the finding that purified TAU readily forms liquid condensate which matures into solid phase over time. Scratches, air bubbles, and distinct microscope coverslip decorations differentially influence droplet maturation, suggesting a catalytic role of surfaces in templating the maturation process which will be examined here by surface modification of glass slide starting from hydrophilic to hydrophobic to super-hydrophobic surface. Another important issue remains unsettled: when and where the solidification starts from within the condensate and whether the chaperones can dissolve the solid phase. We aim to answer these important questions in this work by applying fluorescence microscopy and advanced image processing tool.