Lipid droplets as substrates for protein phase separation.

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

Steric pressure between glycosylated transmembrane proteins inhibits internalization by endocytosis.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids.

The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the standard for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell-cell and cell-matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel®, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clinical translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.

The effects of size and shape of the ovarian cancer spheroids on the drug resistance and migration.

BACKGROUND: High fatality in ovarian cancer is attributed to metastasis, propagated by the release of multi-cellular aggregates/spheroids into the peritoneal cavity and their subsequent mesothelial invasion of peritoneal organs. Spheroids are therefore a common and clinically relevant in vitro model for ovarian cancer research. Spheroids in patients vary significantly in size and shape and display enhanced resistance to anti-cancer drugs compared to monolayers. However, there is no consensus on how spheroid size and shape affect drug resistance. Moreover, existing data regarding the influence of epithelial-to-mesenchymal transition (EMT) profile on spheroid shape and migration is inconclusive. METHODS: We formed spheroids with OVCAR-3 and OVCAR-8 cells, chosen for their established genetic similarity to the patient tumor samples. We monitored their morphology using confocal microscope with dipping objective and fluorescent microscope. We characterized important EMT biomarkers; E-cadherin, Vimentin and Slug through western blotting in monolayers and spheroids. We treated these spheroids with Taxol and Cisplatin and investigated their migratory profile based on their morphology. RESULTS: We report two distinct multicellular structures: loose aggregates (OVCAR-3) and compact spheroids (OVCAR-8). We attribute these different morphologies to the expression of the EMT biomarkers, and their changes upon spheroid formation. Importantly, we did not observe a difference in resistance to the anti-cancer drugs as a function of spheroid size and shape. However, migration capacity of compact spheroid (OVCAR-8) was 15-fold higher compared to that of loose aggregates (OVCAR-3). CONCLUSIONS: These results highlight the importance of spheroid size and shape on anti-cancer drug resistance and migration profiles. The results of this study can, therefore, help to elucidate general rules for ovarian cancer studies based on 3D samples.